Method and materials relating to novel stem cell growth factor-like polypeptides and polynucleotides

ABSTRACT

The invention provides novel polynucleotides and polypeptides encoded by such polynucleotides and mutants or variants thereof that correspond to a novel human stem cell growth factor-like protein. These polynucleotides comprise nucleic acid sequences isolated from cDNA libraries from human testis cells (Hyseq clone identification numbers 2880984 and 2881695), from human fetal skin (Hyseq clone identification number 15375176), adult spleen (Hyseq clone identification number 14856094), and human endothelial cells (Hyseq clone identification numbers 13804756, 13687487, 13804756). Other aspects of the invention include vectors containing processes for producing novel human stem cell growth factor-like polypeptides, and antibodies specific for such polypeptides.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application Ser.No. 60/282,397 filed Apr. 5, 2001, and U.S. Provisional Application Ser.No. 60/215,733, filed Jun. 28, 2000, and U.S. Provisional ApplicationSer. No. 60/266,614 filed Feb. 5, 2001, Attorney Docket No. 21272-039,is a continuation-in-part application of U.S. application Ser. No.09/757,562 filed Jan. 9, 2001 entitled “Methods and Materials Relatingto Novel Stem Cell Growth Factor-Like Polypeptides and Polynucleotides”,Attorney Docket No. HYS-4CON; which in turn is a continuationapplication of U.S. application Ser. No. 09/543,774 filed Apr. 5, 2000entitled “Methods and Materials Relating to Novel Stem Cell GrowthFactor-Like Polypeptides and Polynucleotides”, Attorney Docket No.HYS-4; which in turn is a continuation-in-part application of U.S.application Ser. No. 09/496,914 filed Feb. 3, 2000, entitled “NovelContigs Obtained from Various Libraries”, Attorney Docket No. 787; allof which are incorporated herein by reference in their entirety.

2. TECHNICAL FIELD

The present invention provides novel polynucleotides and proteinsencoded by such polynucleotides, along with uses for thesepolynucleotides and proteins, for example in therapeutic, diagnostic andresearch methods. In particular, the invention relates to a novel humanstem cell growth factor-like protein.

2.1 BACKGROUND ART

Technology aimed at the discovery of protein factors (including e.g.,cytokines, such as lymphokines, interferons, circulating solublefactors, chemokines, and interleukins) has matured rapidly over the pastdecade. The now routine hybridization cloning and expression cloningtechniques clone novel polynucleotides “directly” in the sense that theyrely on information directly related to the discovered protein (i.e.,partial. DNA/amino acid sequence of the protein in the case ofhybridization cloning; activity of the protein in the case of expressioncloning). More recent “indirect” cloning techniques such as signalsequence cloning, which isolates DNA sequences based on the presence ofa now well-recognized secretory leader sequence motif, as well asvarious PCR-based or low stringency hybridization-based cloningtechniques, have advanced the state of the art by making available largenumbers of DNA/amino acid sequences for proteins that are known to havebiological activity, for example, by virtue of their secreted nature inthe case of leader sequence cloning, by virtue of their cell or tissuesource in the case of PCR-based techniques, or by virtue of structuralsimilarity to other genes of known biological activity.

Identified polynucleotide and polypeptide sequences have numerousapplications in, for example, diagnostics, forensics, gene mapping;identification of mutations responsible for genetic disorders or othertraits, to assess biodiversity, and to produce many other types of dataand products dependent on DNA and amino acid sequences.

3. SUMMARY OF THE INVENTION

The present invention provides an isolated polynucleotide encoding apolypeptide having stem cell growth factor activity, said polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 orthe mature protein coding portion thereof, or a fragment, analog,variant or derivative thereof that encodes a polypeptide retaining stemcell growth factor activity. These polypeptides include those whichhybridize to the complement of the nucleotide sequence of SEQ ID NO: 9,11, 12, 31 or 33 under stringent hybridization conditions, those whichcomprise a nucleotide sequence having greater than about 85% sequenceidentity with the nucleotide sequence of SEQ ID NO:9, 11, 12, 31 or 33,those which comprise a nucleotide sequence having greater than about 90%sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12,31 or 33 and those polypeptides which comprise a nucleotide sequencehaving greater than about 92% sequence identity with the nucleotidesequence of SEQ ID NO:9, 11, 12, 31 or 33. 14. The polynucleotides maybe a DNA. The present invention also encompasses polynucleotides whichcomprise the complement of these polynucleotides.

The present invention provides for isolated polynucleotide encoding apolypeptide having stem cell growth factor activity, said polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 orthe mature protein coding portion thereof, or a fragment, analog,variant or derivative thereof that encodes a polypeptide retaining stemcell growth factor activity with the proviso that said polynucleotidesequence does not consist of the nucleotide sequence of SEQ ID NO: 47.These polypeptides include those which hybridize to the complement ofthe nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 understringent hybridization conditions and with the proviso that saidpolynucleotide sequence does not consist of the nucleotide sequence ofSEQ ID NO: 47, those which comprise a nucleotide sequence having greaterthan about 85% sequence identity with the nucleotide sequence of SEQ IDNO:9, 11, 12, 31 or 33 and with the proviso that said polynucleotidesequence does consist of the nucleotide sequence of SEQ ID NO: 47, thosewhich comprise a nucleotide sequence having greater than about 90%sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12,31 or 33 and with the proviso that said polynucleotide sequence does notconsist of the nucleotide sequence of SEQ ID NO: 47, and thosepolypeptides which comprise a nucleotide sequence having greater thanabout 92% sequence identity with the nucleotide sequence of SEQ ID NO:9,11, 12, 31 or 33 and with the proviso that said polynucleotide sequencedoes not consist of the nucleotide sequence of SEQ ID NO: 47.

The present invention provides for an isolated polynucleotide thatcomprises the mature protein coding sequence of SEQ ID NO: 9, 11, 12, 31or 33. Th invention also provides for an isolated polynucleotide thatcomprises the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33.

The invention provides for a DNA encoding a polypeptide having stem cellgrowth factor activity, said polynucleotide comprising the nucleotidesequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein codingportion thereof, or a fragment, analog, variant or derivative thereofthat encodes a polypeptide retaining stem cell growth factor activitywherein the encoded polypeptide has an amino acid sequence comprising atleast amino acid residues 22 to 279 of SEQ ID NO: 32, or an amino acidsequence comprising at least amino acid residues 22 to 272 of SEQ ID NO:34; or the encoded polypeptide has an amino acid sequence includingdeletion, substitution or insertion of one or several amino acids in theamino acid sequence comprising at least amino acid residues 22 to 279 ofSEQ ID NO: 32, or an amino acid sequence comprising at least amino acidresidues 22 to 272 of SEQ ID NO: 34, and which has an activity tosupport proliferation or survival of hematopoietic stem cell orhematopoietic progenitor cell, with a proviso that C-terminal amino acidsequence does not comprise the amino acid sequence of SEQ ID NO: 46.

The invention provides for a DNA encoding a polypeptide having stem cellgrowth factor activity, said polynucleotide comprising the nucleotidesequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein codingportion thereof, or a fragment, analog, variant or derivative thereofthat encodes a polypeptide retaining stem cell growth factor activity,which is a DNA which comprises at least nucleotides 574 to 1347 of SEQID NO: 31; or a DNA which is hybridizable with the nucleotide sequenceof SEQ ID NO: 31 or a probe prepared from said sequence, under stringentconditions, and which has an activity to support proliferation orsurvival of hematopoietic stem cell or hematopoietic progenitor cell.These include DNAs which hybridize under the following stringentconditions: 6×SSC/5× Denhardt, 0.5% SDS and 68° C. (SSC 3M NaCl, 0.3Msodium citrate, 50× Denhardt/1% BSA/1% polyvinyl pyrrolidone, 1% Ficoll400/, or 6×SSC, 5× Denhardt, 0.5% SDS, 50% formamide and 42° C.

The invention provides for a DNA encoding a polynucleotide encoding apolypeptide having stem cell growth factor activity, said polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 orthe mature protein coding portion thereof, or a fragment, analog,variant or derivative thereof that encodes a polypeptide retaining stemcell growth factor activity, which is a DNA which comprises at leastnucleotides 321 to 1074 of SEQ ID NO: 33; or DNA which is hybridizablewith the nucleotide sequence of SEQ ID NO: 33 or a prove prepared fromsaid sequence, under stringent conditions, and which has an activity tosupport proliferation or survival of hematopoietic stem cell orhematopoietic progenitor cell. These include DNAs which hybridize underthe following stringent conditions: 6×SSC/5× Denhardt, 0.5% SDS and 68°C. (SSC 3M NaCl, 0.3M sodium citrate, 50× Denhardt/1% BSA/1% polyvinylpyrrolidone, 1% Ficoll 400, or 6×SSC, 5× Denhardt, 0.5% SDS, 50%Formamide and 42° C.

The invention also provides for vectors, including expression vectors,comprising the polynucleotide of the present invention. The inventionfurther provides for host cells genetically engineered to express apolynucleotide of the present invention. The invention provides for hostcells genetically engineered to contain a polynucleotide of the presentinvention in operative association with a regulatory sequence thatcontrols expression of the polynucleotide in the host cell. These hostcells include those which have been genetically engineered to contain aheterologous regulatory sequence that increases expression of anendogenous polynucleotide.

The invention provides for a method of producing a polypeptide havingstem cell growth factor activity comprising growing these host cells ina culture medium under conditions that permit expression of saidpolypeptide and isolating said polypeptide from said host cell or saidculture medium The invention also encompasses a polypeptide produced bythis method.

The invention provides for an isolated polypeptide comprising the aminoacid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature proteinportion thereof, or a fragment, analog, variant or derivative thereofthat retains stem cell growth factor activity. These polypeptidesinclude polypeptides which are encoded by an isolated polynucleotideencoding a polypeptide having stem cell growth factor activity, saidpolynucleotide comprising the nucleotide sequence of SEQ ID NO: 9, 11,12, 31 or 33 or the mature protein coding portion thereof, or afragment, analog, variant or derivative thereof that encodes apolypeptide retaining stem cell growth factor activity and whichhybridizes to the complement of the nucleotide sequence of SEQ ID NO: 9,11, 12, 31 or 33 under stringent hybridization conditions.

The invention provides for an isolated polypeptide comprising the aminoacid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature proteinportion thereof, or a fragment, analog, variant or derivative thereofthat retains stem cell growth factor activity which comprises an aminoacid sequence having greater than about 85% sequence identity with thenucleotide sequence of SEQ ID NO: 10, 13, 16, 32 or 34, an amino acidsequence having greater than about 92% sequence identity with thenucleotide sequence of SEQ ID NO: 10, 13, 16, 32 or 34, with the provisothat said polypeptide sequence does not consist of the amino acidsequence of SEQ ID NO: 48.

The invention also provides for an isolated polypeptide comprising themature protein portion of SEQ ID NO: 10, 13, 16, 32 or 34.

The invention provides for an isolated polypeptide comprising the aminoacid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature proteinportion thereof, or a fragment, analog, variant or derivative thereofthat retains stem cell growth factor activity, wherein the polypeptidecomprises one or more motifs selected from the group of a laminin-typeEGF-like domain, a membrane attack complex component/perforin domain,and neurohypophysial hormone signature.

The invention provides for polypeptides which are an expression productof a DNA of the present invention, where these polypeptide which have anactivity to support proliferation or survival of hematopoietic stem cellor hematopoietic progenitor cell, with the proviso that the C-terminalamino acid sequence does not comprise the amino acid sequence of SEQ IDNO: 46.

The invention provides for an isolated polynucleotide that comprises thenucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33, which has anamino acid sequence comprising at least amino acid residues 22 to 279 ofSEQ ID NO: 32, or an amino acid sequence including deletion,substitution or insertion of one or several amino acids in the aminoacid sequence comprising at least amino acid residues 22 to 279 of SEQID NO: 32

The invention provides an isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature proteinportion thereof, or a fragment, analog, variant or derivative thereofthat retains stem cell growth factor activity polypeptide, which has anamino acid sequence comprising at least amino acid residues 22 to 272 ofSEQ ID NO: 34, or an amino acid sequence including deletion,substitution or insertion of one or several amino acids in the aminoacid sequence comprising at least amino acid residues 22 to 272 of SEQID NO: 34.

The invention also provides for an isolated polypeptide comprising theamino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the matureprotein portion thereof, or a fragment, analog, variant or derivativethereof that retains stem cell growth factor activity, which is modifiedwith one or more modifying agent selected from the group consisting ofpolyethylene glycol (PEG), dextran, poly(N-vinyl-pyrrolidone),polypropylene glycol homopolymer, copolymer of polypropyleneoxide/ethylene oxide, polyoxyethylated polyol and polyvinyl alcohol.

The invention provides for an isolated polypeptide comprising the aminoacid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature proteinportion thereof, or a fragment, analog, variant or derivative thereofthat retains stem cell growth factor activity which comprises at leastten consecutive amino acids from SEQ ID NO: 10 or 13.

The invention also provides for an isolated polypeptide comprising theamino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the matureprotein portion thereof, or a fragment, analog, variant or derivativethereof that retains stem cell growth factor activity, which comprisesat least ten consecutive amino acids from the C-terminal seventeen aminoacids of SEQ ID NO: 10 or 13.

The invention provides for a polypeptide with biological activity, saidpolypeptide comprising at least 272 amino acids and having at least 98%identity with SEQ ID NO: 10. The invention also provides for an isolatedpolypeptide with stem cell growth factor activity having at least 90%identity with SEQ ID NO: 10, 13, or 16 and lacking amino acid sequenceGIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29) wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine.

The invention also provides for an isolated polypeptide with stem cellgrowth factor activity having at least 90% identity with SEQ ID NO: 10,13, or 16 and lacking any 10 consecutive amino acids from a amino acidsequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29), wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine.

The invention provides for an isolated polypeptide with stem cell growthfactor activity having at least an amino acid sequence SVSVSTVH (SEQ IDNO: 27) or VSVSTVH (SEQ ID NO: 28), wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine.

The invention encompasses a polynucleotide which encodes any of thepolypeptides of the present invention.

The invention provides for a kit comprising an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 10, 13, 16, 32 or 34,or the mature protein portion thereof, or a fragment, analog, variant orderivative thereof that retains stem cell growth factor activity.

The invention further provides for a culture medium comprising an amountof an isolated polypeptide comprising the amino acid sequence of SEQ IDNO: 10, 13, 16, 32 or 34, or the mature protein portion thereof, or afragment, analog, variant or derivative thereof that retains stem cellgrowth factor activity polypeptide, wherein the amount is effective tomaintain survival of or promote proliferation of a stem cell or germcell.

The composition comprising an isolated polypeptide comprising the aminoacid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature proteinportion thereof, or a fragment, analog, variant or derivative thereofthat retains stem cell growth factor activity and a pharmaceuticallyacceptable carrier or diluent. These compositions can be pharmaceuticalcompositions including those having an effect to support proliferationor survival of hematopoietic stem cell or hematopoietic progenitor cell,which comprises: a polypeptide which has an amino acid sequencecomprising at least amino acid residues 22 to 279 of SEQ ID NO: 32, oran amino acid sequence comprising at least amino acid residues 22 to 272of SEQ ID NO: 34; or a polypeptide which has an amino acid sequenceincluding deletion, substitution or insertion of one or several aminoacids in the amino acid sequence comprising at least amino acid residues22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising atleast amino acid residues 22 to 272 of SEQ ID NO: 34, and which has anactivity to support proliferation or survival of hematopoietic stem cellor hematopoietic progenitor cell.

The invention provides for an antibody that binds to an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 10, 13, 16,32 or 34, or the mature protein portion thereof, or a fragment, analog,variant or derivative thereof that retains stem cell growth factoractivity. The antibodies of the present invention may specifically bindsto a polypeptide having the amino acid sequence of SEQ ID NO: 10, 13,16, 32 or 34 including those which do not bind to a polypeptide havingthe amino acid sequence of SEQ ID NO: 48. The antibodies of the presentinvention include polyclonal antibodies, monoclonal antibodies, antibodyfragments, chimeric antibodies, and humanized antibodies. Further, theinvention encompasses kits comprising the antibodies of the presentinvention.

The invention provides for a method for detecting a polynucleotide ofthe present invention in a sample, comprising: a) contacting the samplewith a compound that binds to and forms a complex with thepolynucleotide for a period sufficient to form the complex; and b)detecting the complex, so that if a complex is detected, thepolynucleotide is detected. The invention also provides for methods fordetecting a polynucleotide of the present invention in a sample,comprising: a) contacting the sample under stringent hybridizationconditions with nucleic acid primers that anneal to the polynucleotideunder such conditions; b) amplifying a product comprising at least aportion of the polynucleotide; and c) detecting said product and therebythe polynucleotide in the sample. These methods include a method whereinthe polynucleotide detected encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO: 10, 13, 16, 32 or 34, or the mature proteinportion thereof, or a fragment, analog, variant or derivative thereofthat retains stem cell growth factor activity a polypeptide of claim 23,and the method further comprises reverse transcribing an annealed RNAmolecule into a cDNA polynucleotide.

The invention also provides for a method for detecting a polypeptide ofthe present invnetion in a sample, comprising: a) contacting the samplewith a compound that binds to and forms a complex with the polypeptideunder conditions and for a period sufficient to form the complex; and b)detecting formation of the complex, so that if a complex formation isdetected, the polypeptide is detected.

The invention also provides for a method for identifying a compound thatbinds to a polypeptide of the invention, comprising: a) contacting thecompound with the polypeptide under conditions and for a time sufficientto form a polypeptide/compound complex; and b) detecting the complex, sothat if the polypeptide/compound complex is detected, a compound thatbinds to the polypeptide is identified.

The invention also provides for a method for identifying a compound thatbinds to the polypeptide of the present invention, comprising: a)contacting the compound with the polypeptide, in a cell, for a timesufficient to form a polypeptide/compound complex, wherein the complexdrives expression of a reporter gene sequence in the cell; and b)detecting the complex by detecting reporter gene sequence expression, sothat if the polypeptide/compound complex is detected, a compound thatbinds to the polypeptide is identified.

The invention provides for a nucleic acid array comprising apolynucleotide of the present invention or a unique segment of apolynucleotide of the present invnetion attached to a surface. Thesearrays include those which full-matches to the polynucleotide or aunique segment of the polynucleotide of the present inventions, thosewhich detect mismatches to the polynucleotide or a unique segment of thepolynucleotide of the present invention.

The invention provides for a method of treatment of a subject in need ofenhanced activity or expression of stem cell growth factor-likepolypeptide of the present invention comprising administering to thesubject: (a) a composition comprising a therapeutic amount of an agonistof said polypeptide; (b) a composition comprising a therapeutic amountof the polypeptide; or (c) a composition comprising a therapeutic amountof a polynucleotide encoding the polypeptide in form and underconditions such that the polypeptide is produced; said compositioncomprising a pharmaceutically acceptable carrier or diluent.

The invention also provdies for a method of treatment of a subjecthaving need of decreased activity or expression of stem cell growthfactor-like polypeptide of the present invention comprisingadministering to the subject: (a) a composition comprising a therapeuticamount of an antagonist of said polypeptide; (b) a compositioncomprising a therapeutic amount of the polynucleotide that inhibits theexpression of the nucleotide sequence encoding said polypeptide; and (c)a composition comprising a therapeutic amount of a polypeptide thatcompetes with the stem cell growth factor-like polypeptide for itsligand; said composition comprising a pharmaceutically acceptablecarrier or diluent.

The invention also provides for a method of supporting proliferation orsurvival of a stem cell or germ cell comprising contacting said cellwith an amount of a polypeptide of the present invention effective tomaintain survival of or promote proliferation of said cell. Thesemethods include those wherein said cell is a primordial germ cell, germline stem cell, embryonic stem cell, hematopoietic stem cell,hematopoietic progenitor cell, pluripotent cell, or totipotent cell.These methods also include those wherein the polypeptide comprises anamino acid sequence of SEQ ID NO: 10, 13, or 16, or comprises an aminoacid sequence 90% identical to SEQ ID NO. 10, 13, or 16. These methodsfurther include those wherein the stem cell growth factor-likepolypeptide is encoded by a polynucleotide that hybridizes to thecomplement of a polynucleotide encoding SEQ ID NO: 10, 13, or 16 understringent hybridization conditions.

The invention encompasses a stromal cell genetically engineered toexpress a polypeptide of the invention in an amount effective to supportproliferation or survival of a stem cell or germ cell. These cellsinclude primordial germ cells germ line stem cells embryonic stem cellshematopoietic stem cells hematopoietic progenitor cells pluripotentcells or totipotent cells

The invention provides for an implant comprising a cell geneticallyengineered to express a polypeptide of the present invention in anamount effective to support proliferation or survival of a stem cell orgerm cell. These implants of the present invention include those whereinthe cell is a primordial germ cell, germ line stem cell, embryonic stemcell, hematopoietic stem cell, hematopoietic progenitor cell,pluripotent cell, or totipotent cell.

The invention provides for an isolated polynucleotide comprising theprotein coding cDNA insert of the plasmid deposited with the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology (Zip code 305-8566; Higashi 1-1-3, Tsukuba,Ibaraki, Japan) on Jun. 26, 2000 under accession number FERM BP-7198 andthe mature polypeptide expressed by this polynucleotide of in a suitablehost cell.

The invention also provides for an isolated polynucleotide comprisingthe protein coding cDNA insert of the plasmid deposited with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology (Zip code 305-8566; Higashi 1-1-3,Tsukuba, Ibaraki, Japan) on Jun. 26, 2000 under accession number FERMBP-7197 and the mature polypeptide expression product expressed by thispolynucleotide in a suitable host cell.

Optionally preferred are polynucleotides and polypeptides other than thenucleotide sequence set forth as SEQ ID NO: 3284 (and the polypeptidesequence encoded therein) in U.S. application Ser. No. 09/496,914 filedFeb. 3, 2000 and the protein set out in Genbank Accession No. BAB28811.

The compositions of the present invention include novel isolatedpolypeptides, novel isolated polynucleotides encoding such polypeptides,including recombinant DNA molecules, cloned genes or degenerate variantsthereof, especially naturally occurring variants such as allelicvariants, antisense polynucleotide molecules, and antibodies thatspecifically recognize one or more epitopes present on suchpolypeptides, as well as hybridomas producing such antibodies.Specifically, the polynucleotides of the present invention are based onpolynucleotides isolated from cDNA libraries prepared from human testiscells (Hyseq clone identification numbers 2880984 and 2881695), fromhuman fetal skin (Hyseq clone identification number 15375176), adultspleen (Hyseq clone identification number 14856094), and humanendothelial cells (Hyseq clone identification numbers 13804756,13687487, 13804756).

In one aspect, the invention involves an isolated polynucleotide withstem cell growth factor activity comprising a nucleotide sequence of SEQID NO: 9, 11, 12, 31 or 30, the mature protein coding portion thereof,the extracellular coding portion thereof, or the active domain codingportion thereof.

In one embodiment, the invention involves an isolated polynucleotideencoding a polypeptide with biological activity, and said polynucleotidehybridizes to the complement of the polynucleotide with stem cell growthfactor activity under stringent hybridization conditions.

In a further embodiment, the invention involves an isolatedpolynucleotide encoding a polypeptide with biological activity, saidpolynucleotide having at least about 92% sequence identity with thepolynucleotide with stem cell growth factor activity.

In a further embodiment, the invention involves an isolatedpolynucleotide encoding a polypeptide with biological activity, saidpolypeptide having greater than about 95% sequence identity with thepolynucleotide with stem cell growth factor activity.

In a still further embodiment, the polynucleotide with stem cell growthfactor activity is a DNA.

In another embodiment, the invention involves an isolated polynucleotidewhich comprises the complement of the polynucleotide with stem cellgrowth factor activity.

The invention also involves a vector comprising the polynucleotide withstem cell growth factor activity. Alternatively, the invention involvesan expression vector comprising the polynucleotide with stem cell growthfactor activity. A host cell genetically engineered to express thepolynucleotide with stem cell growth factor activity is also provided.The host cell genetically engineered to contain the polynucleotide withstem cell growth factor activity in operative association with aregulatory sequence that controls expression of the polynucleotide inthe host cell.

In another aspect, the invention involves an isolated polypeptidecomprising an amino acid sequence consisting of SEQ ID NO: 10, 13, 16,32 or 34, the mature protein portion thereof, the extracellular portionthereof, or active domain thereof.

Also provided is a composition comprising the polypeptide and a carrier.In another embodiment, the invention involves an antibody directedagainst the polypeptide.

In another aspect, the invention involves a method for detecting thepolynucleotide with stem cell growth factor activity in a sample,comprising contacting the sample with a compound that binds to and formsa complex with the polynucleotide for a period sufficient to form thecomplex; and detecting the complex, so that if a complex is detected,the polynucleotide is detected.

The invention also involves a method for detecting the polynucleotidewith stem cell growth factor activity in a sample, comprising contactingthe sample under stringent hybridization conditions with nucleic acidprimers that anneal to the polynucleotide under such conditions;amplifying a product comprising at least a portion of thepolynucleotide; and detecting said product and thereby thepolynucleotide in the sample.

In a further embodiment, the polynucleotide is an RNA molecule thatencodes the polypeptide, and the method further comprises reversetranscribing an annealed RNA molecule into a cDNA polynucleotide.

Also provided is a method for detecting the polypeptide in a sample,comprising contacting the sample with a compound that binds to and formsa complex with the polypeptide under conditions and for a periodsufficient to form the complex; and detecting formation of the complex,so that if a complex formation is detected, the polypeptide is detected.

In another embodiment, the invention provides a method for identifying acompound that binds to the polypeptide, comprising contacting thecompound with the polypeptide of under conditions and for a timesufficient to form a polypeptide/compound complex; and detecting thecomplex, so that if the polypeptide/compound complex is detected, acompound that binds to the polypeptide is identified.

In a further embodiment, the invention involves a method for identifyinga compound that binds to the polypeptide, comprising contacting thecompound with the polypeptide in a cell, for a time sufficient to form apolypeptide/compound complex, wherein the complex drives expression of areporter gene sequence in the cell; and detecting the complex bydetecting reporter gene sequence expression, so that if thepolypeptide/compound complex is detected, a compound that binds to thepolypeptide is identified.

In another embodiment, the invention involves a method of producing thepolypeptide, comprising, culturing the host cell for a period of timesufficient to express the polypeptide in said cell; and isolating thepolypeptide from the cell culture or cells.

In another aspect, the invention involves a kit comprising thepolypeptide. Also provided is a nucleic acid array comprising thepolynucleotide or a segment of the polynucleotide attached to a surface.In a further embodiment, the array detects full-matches to thepolynucleotide or a unique segment of the polynucleotide. In anotherembodiment, the array detects mismatches to the polynucleotide or aunique segment of the polynucleotide.

The invention also provides for a method of treatment of a subject inneed thereof enhanced activity or expression of stem cell growthfactor-like polypeptide comprising administering to the subject acomposition selected from the group consisting of a) therapeutic amountof an agonist of said polypeptide; b) a therapeutic amount of thepolypeptide; and c) a therapeutic amount of a polynucleotide encodingthe polypeptide in form and under conditions such that the polypeptideis produced, and a pharmaceutically acceptable carrier. The inventionalso provides for a method of treatment of a subject having need toinhibit activity or expression of stem cell growth factor-likepolypeptide comprising administering to the subject a compositionselected from the group consisting of a) a therapeutic amount of anantagonist of said polypeptide; b) a therapeutic amount of thepolynucleotide that inhibits the expression of the nucleotide sequenceencoding said polypeptide; and c) a therapeutic amount of a polypeptidethat competes with the stem cell growth factor-like polypeptide for itsligand, and a pharmaceutically acceptable carrier.

In another embodiment, the invention involves a polypeptide having stemcell growth factor activity comprising at least ten consecutive aminoacids from SEQ ID NO: 10, 13, 16, 32 or 34. In still another embodiment,the invention involves this polypeptide comprising at least tenconsecutive amino acids from the C-terminal seventeen amino acids of SEQID NO: 10, 13, 16, 32 or 34.

Also provided is a polypeptide with biological activity, saidpolypeptide comprising at least 272 amino acids and having at least 98%identity with SEQ ID NO: 10 or 34 or said polypeptide comprising atleast 273 amino acids and having at least 98% identity with SEQ ID NO:13.

In a further embodiment, the invention involves an isolated polypeptidewith stem cell growth factor activity having at least 90% identity withSEQ ID NO: 10, 13, 16, 32 or 34 and lacking amino acid sequenceGIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29) wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine.

In yet another embodiment, the invention involves an isolatedpolypeptide with stem cell growth factor activity having at least 90%identity with SEQ ID NO: 10, 13, 16, 32 or 34 and lacking any 10consecutive amino acids from amino acid sequenceGIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29), wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine.

In another embodiment, the invention concerns a method of maintaining orpromoting proliferation of a cell selected from the group consisting ofprimordial germ cells, germ line stem cells, embryonic stem cells,pluripotent cell, and totipotent cells, comprising contacting the cellwith an effective amount of stem cell growth factor-like polypeptide. Ina further embodiment, the polypeptide comprises an amino acid sequenceof SEQ ID NO: 10, 13, 16, 32 or 34, or comprises an amino acid sequence90% identical to SEQ ID NO. 10, 13, 16, 32 or 34. In still a furtherembodiment, the stem cell growth factor-like polypeptide is encoded by apolynucleotide that hybridizes to the complement of a polynucleotideencoding SEQ ID NO: 10, 13, 16, 32 or 34 under stringent hybridizationconditions.

The invention also involves an isolated polypeptide with stem cellgrowth factor activity having at least an amino acid sequence SVSVSTVH(SEQ ID NO: 27) or VSVSTVH (SEQ ID NO: 28), wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine.

In an additional embodiment, the invention concerns the polypeptideaccording to this invention, wherein the polypeptide comprises one ormore motifs selected from the group of a laminin-type EGF-like domain, amembrane attack complex component/perforin domain, and neurohypophysialhormone signature.

The invention also encompasses any polynucleotides encoding apolypeptide according to this invention.

The compositions of the present invention additionally include vectors,including expression vectors, containing the polynucleotides of theinvention, cells genetically engineered to contain such polynucleotidesand cells genetically engineered to express such polynucleotides.

The isolated polynucleotides of the invention include, but are notlimited to, a polynucleotide comprising any one of the nucleotidesequences set forth in the SEQ ID NO: 9, 11, 12, 31 or 33; apolynucleotide comprising any of the full length protein codingsequences of the SEQ ID NO: 9, 11, 12, 31 or 33; and a polynucleotidecomprising any of the nucleotide sequences of the mature protein codingsequences of the SEQ ID NO: 9, 11, 12, 31 or 33. The polynucleotides ofthe present invention also include, but are not limited to, apolynucleotide that hybridizes under stringent hybridization conditionsto (a) the complement of any one of the nucleotide sequences set forthin the SEQ ID NO: 9, 11, 12, 31 or 33; (b) a nucleotide sequenceencoding SEQ ID NO: 10, 13-24, 32 or 34; a polynucleotide which is anallelic variant of any polynucleotides recited above; a polynucleotidewhich encodes a species homolog (e.g. orthologs) of any of the proteinsrecited above; or a polynucleotide that encodes a polypeptide comprisinga specific domain or truncation of any of the polypeptides comprisingSEQ ID NO: 10, 13-24, 32 or 34.

The nucleic acid sequences of the present invention also include thesequence information from the nucleic acid sequences of SEQ ID NO: 11,12, 31 or 33. The sequence information can be a segment of any one ofSEQ ID NO: 1-7 that uniquely identifies or represents the sequenceinformation of SEQ ID NO: 11, 12, 31 or 33. One such segment can be atwenty-mer nucleic acid sequence because the probability that atwenty-mer is fully matched in the human genome is 1 in 300. In thehuman genome, there are three billion base pairs in one set ofchromosome. Because there are 4²⁰ possible twenty-mers exist, there are300 times more twenty-mers than there are base pairs in a set of humanchromosome. Using the same analysis, the probability for a seventeen-merto be fully matched in the human genome is approximately 1 in 5. Whenthese segments are used in arrays for expression studies, fifteen-mersegment can be used. The probability that the fifteen-mer is fullymatched in the expressed sequences is also approximately one in fivebecause expressed sequences in one tissue comprise approximately 5% ofthe entire genome sequence. Preferably, the nucleic acid fragment orsubsequence comprise the twenty-one 3′ coding nucleotides.

Similarly, when using sequence information for detecting a singlemismatch, a segment can be a twenty-five mer. The probability that thetwenty-five mer would appear in a human genome with a single mismatch iscalculated by multiplying the probability for a full match (1÷4²⁴) timesthe increased probability for mismatch at each nucleotide position(3×25). The probability that an eighteen mer with a single mismatch canbe detected in an array for expression studies is approximately one infive. The probability that a twenty-mer with a single mismatch can bedetected in a human genome is approximately one in five.

A collection as used in this application can be a collection of only onepolynucleotide. The collection of sequence information or uniqueidentifying information of each sequence can be provided on a nucleicacid array. In one embodiment, segments of sequence information areprovided on a nucleic acid array to detect the polynucleotide thatcontains the segment. The array can be designed to detect full-match ormismatch to the polynucleotide that contains the segment. The collectioncan also be provided in a computer-readable format.

This invention also includes the reverse or direct complement of any ofthe nucleic acid sequences recited above; cloning or expression vectorscontaining the nucleic acid sequences; and host cells or organismstransformed with these expression vectors.

Human stem cell growth factor-like polypeptide (SEQ ID NO: 10 or 34) isapproximately a 272-amino acid protein with a predicted molecular massof approximately 30 kDa unglycosylated. The mouse homolog is set out inSEQ ID. NO:32. Protein database searches with the BLAST algorithmindicate that SEQ ID NO: 10 is homologous to Mus musculus thrombospondintype 1 domain. FIG. 1 shows the alignment of polynucleotide SEQ ID NO: 9and EST sequences SEQ ID NO: 1-7. The sequences of the present invention(SEQ ID NO: 1-12) are expected to have stem cell growth factor activity,including hematopoietic stem cell growth factor activity, as describedherein.

Stem cell growth factor-like polypeptide (SEQ ID NO: 10) also has thefollowing motifs at the designated amino acid sequence corresponding toSEQ ID NO: 10 wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine:

Laminin-type EGF-like (LE) domain proteins at 100ADCDTCFNKNFCTKCKSGFYLHL 122 (SEQ ID NO: 17)

Vertebrate metallothioneins proteins at (SEQ ID NO: 18) 92INKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEANN 137

Endogenous opioids neuropeptides precursors proteins at 33MHPNVSQGCQGGCATCSDYN 52 (SEQ ID NO: 19)

Membrane attack complex components/perforin proteins at (SEQ ID NO: 20)145 IVHCEVSEWNPWSPCTKKGKTCGFKRGTETRVREIIQ 181

HMG-I and HMG-Y DNA-binding domain proteins (Ahook) at 213 KKGRERKRKK222 (SEQ ID NO: 21)

HMG1/2 proteins at (SEQ ID NO: 22) 198KCTVQRKKCQKGERGKKGRERKRKKPNKGESKEAIPDSKSLE 239

VERTEBRATE METALLOTHIONEIN SIGNATURE at 104 TCFNKNFCTKCKSG 117 (SEQ IDNO: 23)

NEUROHYPOPHYSIAL HORMONE SIGNATURE at 148 CEVSEWNPWSPCTKKGKTCG 167 (SEQID NO: 24)

Motif 100-122, a laminin-type EGF-like domain, is a component ofextracellular matrix which promotes cell growth. The membrane attackcomplex component/perforin domain (145-185) is postulated to mediatecell-cell interaction and thus cell growth and differentiation.Neurohypophysial hormone is itself regulated by many other factorsincluding Interleukin-1 beta and Interleukin-6. The presence of thesemotifs are expected in stem cell growth factor activity.

Stem cell growth factor-like protein and/or fragments or derivativeswould have similar activity to stem cell growth factors and anabolicgrowth factors and receptors.

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising SEQ ID NO: 10, 13-24, 32 or 34; or thecorresponding full length or mature protein. Polypeptides of theinvention also include polypeptides with biological activity that areencoded by (a) any of the polynucleotides having a nucleotide sequenceset forth in the SEQ ID NO: 1-9; 11, 12, 31 or 33 or (b) polynucleotidesthat hybridize to the complement of the polynucleotides of (a) understringent hybridization conditions. Biologically or immunologicallyactive variants of any of the protein sequences listed as SEQ ID NO: 10,13-24, 32 or 34, and “substantial equivalents” thereof (e.g., with atleast about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% amino acid sequence identity) that preferably retainbiological activity are also contemplated. The polypeptides of theinvention may be wholly or partially chemically synthesized but arepreferably produced by recombinant means using the geneticallyengineered cells (e.g. host cells) of the invention.

The invention also provides compositions comprising a polypeptide of theinvention. Polypeptide compositions of the invention may furthercomprise an acceptable carrier, such as a hydrophilic, e.g.,pharmaceutically acceptable, carrier.

The invention also provides host cells transformed or transfected with apolynucleotide of the invention.

The invention also relates to methods for producing a polypeptide of theinvention comprising growing a culture of the host cells of theinvention in a suitable culture medium under conditions permittingexpression of the desired polypeptide, and purifying the protein fromthe culture or from the host cells. Preferred embodiments include thosein which the protein produced by such process is a mature form of theprotein.

Polynucleotides according to the invention have numerous applications ina variety of techniques known to those skilled in the art of molecularbiology. These techniques include use as hybridization probes, use asoligomers, or primers, for PCR, use in an array, use incomputer-readable media, use for chromosome and gene mapping, use in therecombinant production of protein, and use in generation of anti-senseDNA or RNA, their chemical analogs and the like. For example, when theexpression of an mRNA is largely restricted to a particular cell ortissue type, polynucleotides of the invention can be used ashybridization probes to detect the presence of the particular cell ortissue mRNA in a sample using, e.g., in situ hybridization.

In other exemplary embodiments, the polynucleotides are used indiagnostics as expressed sequence tags for identifying expressed genesor, as well known in the art and exemplified by Vollrath et al., Science258:52-59 (1992), as expressed sequence tags for physical mapping of thehuman genome.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook, J., et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polypeptides include an assortment of vectors,e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and thelike, that are well known in the art. Accordingly, the invention alsoprovides a vector including a polynucleotide of the invention and a hostcell containing the polynucleotide. In general, the vector contains anorigin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The polypeptides according to the invention can be used in a variety ofconventional procedures and methods that are currently applied to otherproteins. For example, a polypeptide of the invention can be used togenerate an antibody that specifically binds the polypeptide. Suchantibodies, particularly monoclonal antibodies, are useful for detectingor quantitating the polypeptide in tissue. The polypeptides of theinvention can also be used as molecular weight markers, and as a foodsupplement.

Methods are also provided for preventing, treating, or ameliorating amedical condition which comprises the step of administering to amammalian subject a therapeutically effective amount of a compositioncomprising a protein of the present invention and a pharmaceuticallyacceptable carrier.

In particular, the polypeptides and polynucleotides of the invention canbe utilized, for example, as part of methods for the prevention and/ortreatment of disorders involving aberrant protein expression orbiological activity.

The methods of the invention also provides methods for the treatment ofdisorders as recited herein which may involve the administration of thepolynucleotides or polypeptides of the invention to individualsexhibiting symptoms or tendencies related to disorders as recitedherein. In addition, the invention encompasses methods for treatingdiseases or disorders as recited herein comprising the step ofadministering compounds and other substances that modulate the overallactivity of the target gene products. Compounds and other substances caneffect such modulation either on the level of target gene/proteinexpression or target protein activity. Specifically, methods areprovided for preventing, treating or ameliorating a medical condition,including neurological diseases, which comprises administering to amammalian subject, including but not limited to humans, atherapeutically effective amount of a composition comprising apolypeptide of the invention or a therapeutically effective amount of acomposition comprising a binding partner of (e.g., antibody specificallyreactive for) stem cell growth factor-like polypeptides of theinvention. The mechanics of the particular condition or pathology willdictate whether the polypeptides of the invention or binding partners(or inhibitors) of these would be beneficial to the individual in needof treatment.

The invention also provides a method of promoting wound healingcomprising administering a stem cell growth factor-like polypeptide ofthe present invention to the site of a wound or injury. The inventionprovides a method of promoting cell growth and morphogenesis comprisingadministering a stem cell growth factor-like polypeptide of the presentinvention to a medium of nerve cells. According to this method,polypeptides of the invention can be administered to produce an in vitroor in vivo promotion of cellular function. A polypeptide of theinvention can be administered in vivo as a stem cell growth factor aloneor as an adjunct to other therapies.

The invention further provides methods for manufacturing medicamentsuseful in the above described methods.

The present invention further relates to methods for detecting thepresence of the polynucleotides or polypeptides of the invention in asample (e.g., tissue or sample). Such methods can, for example, beutilized as part of prognostic and diagnostic evaluation of disorders asrecited herein and for the identification of subjects exhibiting apredisposition to such conditions. The invention also provides kitscomprising polynucleotide probes and/or monoclonal antibodies, andoptionally quantitative standards, for carrying out methods of theinvention. Furthermore, the invention provides methods for evaluatingthe efficacy of drugs, and monitoring the progress of patients, involvedin clinical trials for the treatment of disorders as recited above.

The invention also provides methods for the identification of compoundsthat modulate (i.e., increase or decrease) the expression or activity ofthe polynucleotides and/or polypeptides of the invention. Such methodscan be utilized, for example, for the identification of compounds thatcan ameliorate symptoms of disorders as recited herein. Such methods caninclude, but are not limited to, assays for identifying compounds andother substances that interact with (e.g., bind to) the polypeptides ofthe invention.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the alignment of SEQ ID NO. 9 with SEQ ID NO. 1-7.

FIG. 2 shows the BLASTP amino acid sequence alignment between the SEQ IDNO: 10, stem cell growth factor-like polypeptide and mousethrombospondin type 1 domain protein SEQ ID NO: 25, indicating that thetwo sequences share 64% similarity over amino acid residues 19-254 ofSEQ ID NO: 10 and 47% identity over the same amino acid residues 19-254of SEQ ID NO: 10, wherein A=Alanine, C=Cysteine, D=Aspartic Acid,E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine,K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

FIG. 3 shows the BLASTP amino acid sequence alignment between the SEQ IDNO: 10, stem cell growth factor-like polypeptide and human secretedprotein clone da228_(—)6 protein (Patent Application No. WO98/49302),SEQ ID NO: 26, indicating that the two sequences share 100% similarityover amino acid residues 1-265 of SEQ ID NO: 10 and 100% identity overthe same amino acid residues 1-265 of SEQ ID NO: 10, wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine. Gaps are presented as dashes.

FIG. 4 shows proliferation statuses of hematopoietic stem cells andhematopoietic progenitor cells determined by a clonogenic assay afterco-culture of CD34 positive hematopoietic stem cells with AGM-s3subclone A9, A7, or D11 cells for two weeks;

FIG. 5 shows proliferation statuses of hematopoietic stem cells andhematopoietic progenitor cells determined by a clonogenic assay afterco-culture of CD34 positive hematopoietic stem cells with AGM-s3subclone A9, A7, or OP9 cells for two weeks;

FIG. 6 shows time course of donor derived lymphoid lineage cells ormyeloid lineage cells reconstitution in irradiated recipient mice thatreceived the hematopoietic stem cells co-cultured with stromal cells;and

FIG. 7 shows time course of donor derived lymphoid lineage cells ormyeloid lineage cells reconstitution in irradiated recipient mice thatreceived the hematopoietic stem cells co-cultured with AGM-s3-A7 celllines (A7/pMXIG-SCR-1 and A7/pMXIG) transfected with a vector includingSCR-1 (pMXIG-SCR-1) or a vector which does not include SCR-1 (pMXIG).

5. DETAILED DESCRIPTION OF THE INVENTION

5.1 Definitions

The term “primordial germ c ells (PGCs)” refers to a small population ofcells set aside from other cell lineages particularly from the yolk sac,mesenteries, or gonadal ridges during embryogenesis that have thepotential to differentiate in to germ cells and other cells. PGCs arethe source from which GSCs and ES cells are derived

The term “germ line stem cells (GSCs)” refers to stem cells derived fromprimordial stem cells that provide a steady and continuous source ofgerm cells for the production of gametes.

The term “embryonic stem cells (ES)” refers to a cell which can giverise to many differentiated cell types in an embryo or an adult,including the germ cells. The PGCs, the GSCs and the ES cells arecapable of self-renewal. Thus these cells not only populate the germline and give rise to a plurality of terminally differentiated cellswhich comprise the adult specialized organs, but are able to regeneratethemselves.

The term “totipotent” refers to the capability of a cell todifferentiate into all of the cell types of an adult organism.

The term “pluripotent” refers to the capability of a cell todifferentiate into a number of differentiated cell types that arepresent in an adult organism. A pluripotent cell is restricted in itsdifferentiation capability in comparison to a totipotent cell.

The term “nucleotide sequence” refers to a heteropolymer of nucleotidesor the sequence of these nucleotides. The terms “nucleic acid” and“polynucleotide” are also used interchangeably herein to refer to aheteropolymer of nucleotides. Generally, nucleic acid segments providedby this invention may be assembled from fragments of the genome andshort oligonucleotide linkers, or from a series of oligonucleotides, orfrom individual nucleotides, to provide a synthetic nucleic acid whichis capable of being expressed in a recombinant transcriptional unitcomprising regulatory elements derived from a microbial or viral operon,or a eukaryotic gene.

The terms “oligonucleotide fragment” or a “polynucleotide fragment”,“portion,” or “segment” is a sequence of nucleotide residues which islong enough to use in polymerase chain reaction (PCR) or varioushybridization procedures to identify or amplify identical or relatedparts of mRNA or DNA molecules. A fragment or segment may uniquelyidentify each polynucleotide sequence of the present invention.

The terms “oligonucleotides” or “nucleic acid probes” are prepared basedon the polynucleotide sequences provided in the present invention.Oligonucleotides comprise portions of such a polynucleotide sequencehaving at least about 15 nucleotides and usually at least about 20nucleotides. Nucleic acid probes comprise portions of such apolynucleotide sequence having fewer nucleotides than about 6 kb,usually fewer than about 1 kb. After appropriate testing to eliminatefalse positives, these probes may, for example, be used to determinewhether specific mRNA molecules are present in a cell or tissue or toisolate similar nucleic acid sequences from chromosomal DNA as describedby Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).

The term “probes” includes naturally occurring or recombinant orchemically synthesized single- or double-stranded nucleic acids. Theymay be labeled by nick translation, Klenow fill-in reaction, PCR, orother methods well known in the art. Probes of the present invention,their preparation and/or labeling are elaborated in Sambrook, J. et al.,1989, Molecular Cloning: A. Laboratory Manual, Cold Spring HarborLaboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols inMolecular Biology, John Wiley & Sons, New York N.Y., both of which areincorporated herein by reference in their entirety.

The term “stringent” is used to refer to conditions that are commonlyunderstood in the art as stringent. Stringent conditions can includehighly stringent conditions (i.e., hybridization to filter-bound DNA in0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringentconditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.). Otherexemplary hybridization conditions are described herein in the examples.

In instances of hybridization of deoxyoligonucleotides, additionalexemplary stringent hybridization conditions include washing in6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C.(for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for23-base oligos).

The term “recombinant,” when used herein to refer to a polypeptide orprotein, means that a polypeptide or protein is derived from recombinant(e.g., microbial, insect, or mammalian) expression systems. “Microbial”refers to recombinant polypeptides or proteins made in bacterial orfungal (e.g., yeast) expression systems. As a product, “recombinantmicrobial” defines a polypeptide or protein essentially free of nativeendogenous substances and unaccompanied by associated nativeglycosylation. Polypeptides or proteins expressed in most bacterialcultures, e.g., E. coli, will be free of glycosylation modifications;polypeptides or proteins expressed in yeast will have a glycosylationpattern in general different from those expressed in mammalian cells.

The term “recombinant expression vehicle or vector” refers to a plasmidor phage or virus or vector, for expressing a polypeptide from a DNA(RNA) sequence. An expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an amino terminalmethionine residue. This residue may or may not be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems as defined herein will expressheterologous polypeptides or proteins upon induction of the regulatoryelements linked to the DNA segment or synthetic gene to be expressed.This term also means host cells which have stably integrated arecombinant genetic element or elements having a regulatory role in geneexpression, for example, promoters or enhancers. Recombinant expressionsystems as defined herein will express polypeptides or proteinsendogenous to the cell upon induction of the regulatory elements linkedto the endogenous DNA segment or gene to be expressed. The cells can beprokaryotic or eukaryotic.

The term “open reading frame,” ORF, means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

The term “expression modulating fragment,” EMF, means a series ofnucleotides which modulates the expression of an operably linked ORF oranother EMF.

As used herein, a sequence is said to “modulate the expression of anoperably linked sequence” when the expression of the sequence is alteredby the presence of the EMF. EMFs include, but are not limited to,promoters, and promoter modulating sequences (inducible elements). Oneclass of EMFs are fragments which induce the expression or an operablylinked ORF in response to a specific regulatory factor or physiologicalevent.

As used herein, an “uptake modulating fragment,” UMF, means a series ofnucleotides which mediate the uptake of a linked DNA fragment into acell. UMFs can be readily identified using known UMFs as a targetsequence or target motif with the computer-based systems describedbelow.

The presence and activity of a UMF can be confirmed by attaching thesuspected UMF to a marker sequence. The resulting nucleic acid moleculeis then incubated with an appropriate host under appropriate conditionsand the uptake of the marker sequence is determined. As described above,a UMF will increase the frequency of uptake of a linked marker sequence.

The term “active” refers to those forms of the polypeptide which retainthe biologic and/or immunologic activities of any naturally occurringpolypeptide. According to the invention, the term “biologically active”means that the polypeptide retains at least one of the biologicalactivities of the polypeptide of the invention. The term. “stem c ellgrowth factor activity” or “stem cell growth factor-like activity”refers to biological activity that is similar to the biological activityof stem cell growth factor polypeptide, such as cell growth ormorphogenesis activity.

The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

The term “derivative” refers to polypeptides chemically modified by suchtechniques as ubiquitination, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins.

The term “variant” (or “analog”) refers to any polypeptide differingfrom naturally occurring polypeptides by amino acid insertions,deletions, and substitutions, created using, e g., recombinant DNAtechniques. Guidance in determining which amino acid residues may bereplaced, added or deleted without abolishing activities of interest,may be found by comparing the sequence of the particular polypeptidewith that of homologous peptides and minimizing the number of amino acidsequence changes made in regions of high homology (conserved regions) orby replacing amino acids with consensus sequence.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements.“Conservative” amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Insertions” or “deletions” are typically in the range ofabout 1 to 5 amino acids. The variation allowed may be experimentallydetermined by systematically making insertions, deletions, orsubstitutions of amino acids in a polypeptide molecule using recombinantDNA techniques and assaying the resulting recombinant variants foractivity.

Alternatively, where alteration of function is desired, insertions,deletions or non-conservative alterations can be engineered to producealtered polypeptides. Such alterations can, for example, alter one ormore of the biological functions or biochemical characteristics of thepolypeptides of the invention. For example, such alterations may changepolypeptide characteristics such as ligand-binding affinities,interchain affinities, or degradation/turnover rate. Further, suchalterations can be selected so as to generate polypeptides that arebetter suited for expression, scale up and the like in the host cellschosen for expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

As used herein, “substantially equivalent” or “substantially similar”can refer both to nucleotide and amino acid sequences, for example amutant sequence, that varies from a reference sequence by one or moresubstitutions, deletions, or additions, the net effect of which does notresult in an adverse functional dissimilarity between the reference andsubject sequences. Typically, such a substantially equivalent sequencevaries from one of those listed herein by no more than about 35% (i.e.,the number of individual residue substitutions, additions, and/ordeletions in a substantially equivalent sequence, as compared to thecorresponding reference sequence, divided by the total number ofresidues in the substantially equivalent sequence is about 0.35 orless). Such a sequence is said to have 65% sequence identity to thelisted sequence. In one embodiment, a substantially equivalent, e.g.,mutant, sequence of the invention varies from a listed sequence by nomore than 30% (70% sequence identity); in a variation of thisembodiment, by no more than 25% (75% sequence identity); and in afurther variation of this embodiment, by no more than 20% (80% sequenceidentity) and in a further variation of this embodiment, by no more than10% (90% sequence identity) and in a further variation of thisembodiment, by no more that 5% (95% sequence identity). Substantiallyequivalent, e.g., mutant, amino acid sequences according to theinvention preferably have at least 80% sequence identity with a listedamino acid sequence, more preferably at least 85% sequence identity,more preferably at least 90%-sequence identity, more preferably at least95% sequence identity, more preferably at least 98% sequence identity,and most preferably at least 99% sequence identity. Substantiallyequivalent nucleotide sequence of the invention can have lower percentsequence identities, taking into account, for example, the redundancy ordegeneracy of the genetic code. Preferably, the nucleotide sequence hasat least about 65% identity, more preferably at least about 75%identity, more preferably at least about 80% sequence identity, morepreferably at least 85% sequence identity, more preferably at least 90%sequence identity, more preferably at least about 95% sequence identity,more preferably at least 98% sequence identity, and most preferably atleast 99% sequence identity. For the purposes of the present invention,sequences having substantially equivalent biological activity andsubstantially equivalent expression characteristics are consideredsubstantially equivalent. For the purposes of determining equivalence,truncation of the mature sequence (e.g., via a mutation which creates aspurious stop codon) should be disregarded. Sequence identity may bedetermined, e.g., using the Jotun Hein method (Hein, J. (1990) MethodsEnzymol. 183:626-645). Identity between sequences can also be determinedby other methods known in the art, e.g. by varying hybridizationconditions.

Nucleic acid sequences encoding such substantially equivalent sequences,e.g., sequences of the recited percent identities can routinely beisolated and identified via standard hybridization procedures well knownto those of skill in the art.

Where desired, an expression vector may be designed to contain a “signalor leader sequence” which will direct the polypeptide through themembrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

A polypeptide “fragment,” “portion,” or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, and, invarious embodiments, at least about 17 or more amino acids. To beactive, any polypeptide must have sufficient length to displaybiological and/or immunological activity.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

The term “activated” cells as used in this application are those whichare engaged in extracellular or intracellular membrane trafficking,including the export of secretory or enzymatic molecules as part of anormal or disease process.

The term “purified” as used herein denotes that the indicated nucleicacid or polypeptide is present in the substantial absence of otherbiological macromolecules, e.g., polynucleotides, proteins, and thelike. In one embodiment, the polynucleotide or polypeptide is purifiedsuch that it constitutes at least 95% by weight, more preferably atleast 99.8% by weight, of the indicated biological macromoleculespresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 1000 daltons, can bepresent).

The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother component normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

The term “infection” refers to the introduction of nucleic acids into asuitable host cell by use of a virus or viral vector.

The term “transformation” means introducing DNA into a suitable hostcell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration.

The term “transfection” refers to the taking up of an expression vectorby a suitable host cell, whether or not any coding sequences are in factexpressed.

The term “intermediate fragment” means a nucleic acid between 5 and 1000bases in length, and preferably between 10 and 40 bp in length.

The term “secreted” includes a protein that is transported across orthrough a membrane, including transport as a result of signal sequencesin its amino acid sequence when it is expressed in a suitable host cell.“Secreted” proteins include without limitation proteins secreted wholly(e.g., soluble proteins) or partially (e.g., receptors) from the cell inwhich they are expressed. “Secreted” proteins also include withoutlimitation proteins which are transported across the membrane of theendoplasmic reticulum. “Secreted” proteins are also intended to includeproteins containing non-typical signal sequences (e.g. Interleukin-1Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143)and factors released from damaged cells (e.g. Interleukin-1 ReceptorAntagonist, see Arend, W. P. et. al. (1998) Annu. Rev. Immunol.16:27-55)

Each of the above terms is meant to encompasses all that is describedfor each, unless the context dictates otherwise.

5.1.1 Description of the Invention

Since stromal cells can support the proliferation or the survival ofhematopoietic stem cells or hematopoietic progenitor cells ex vivo,stromal cells are expected to produce factors mediating supportproliferation or survival of hematopoietic stem cells or hematopoieticprogenitor cells, as defined herein.

An object of the present invention is to provide a factors supportingproliferation or survival of hematopoietic stem cells or hematopoieticprogenitor cells and these factors are/can be derived from the stromalcells.

Mouse stromal cells produce factors supporting the proliferation or thesurvival of hematopoietic stem cells or hematopoietic progenitor cells,as mentioned above. Attention is given that there are two kinds ofstromal cells. One has an ability to support the proliferation orsurvival of hematopoietic stem cells or hematopoietic progenitor cells(hereafter sometimes referred to as “activity to support hematopoieticstem cells”). The other does not have the activity to supporthematopoietic stem cells. This difference in the abilities maybe due todifferential expression of the factors that facilitate supportinghematopoietic stem cells or progenitor cells at the transcription level.That is to say it is speculated that the supportive stromal cellsexpress at high levels of mRNAs coding the factors and thatnon-supportive stromal cells express less mRNAs. Thus mRNAs that codefor the factors maybe among the genes expressed higher in the supportivecells compared to in the non-supportive cells. In this context, theinventors confirmed the hematopoietic stem and/or progenitor cellsupporting ability of AGM-s3-A9, AGM-s3-D11, OP9, and SWISS3T3 celllines and the non-supportive ability of AGM-s3-A7, AGM-s3-G1, and NIH3T3cell lines (AGM-s3-A9, AGM-s3-D11, AGM-s3-A7, and AGM-s3-G1 cell linesare obtained by subcloning the stromal cell strain AGM-s3 derived fromAGM disclosed in the prior application-WO99/03980). Next, the genes thatare highly expressed in AGM-s3-A9, AGM-s3-D11, OP9, and 3T3Swiss celllines and show low expression or are undetected in AGM-s3-A7, AGM-s3-G1,and NIH3T3 cell lines were identified. After the assessment of theabilities of supporting the proliferation or the survival of thehematopoietic stem cells or the hematopoietic progenitor cells of thesegene groups and careful examinations, the present invention has beencompleted.

That is, the present invention provides the followings.

-   (1) A DNA coding for a polypeptide as defined in the following (A)    or (B):    -   (A) a polypeptide which has an amino acid sequence comprising at        least amino acid residues 22 to 279 of SEQ ID NO: 32, or an        amino acid sequence comprising at least amino acid residues 22        to 272 of SEQ ID NO: 34; or    -   (B) a polypeptide which has an amino acid sequence including        deletion, substitution or insertion of one or several amino        acids in the amino acid sequence comprising at least amino acid        residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence        comprising at least amino acid residues 22 to 272 of SEQ ID NO:        34, and which has an activity to support proliferation or        survival of hematopoietic stem cell or hematopoietic progenitor        cell, with a proviso that C-terminal amino acid sequence dose        not comprise the amino acid sequence of SEQ ID NO: 45.-   (2) The DNA according to (1), which is a DNA as defined in the    following (a) or (b):    -   (a) a DNA which comprises at least nucleotides 574 to 1347 of        SEQ ID NO: 31; or    -   (b) a DNA which is hybridizable with the nucleotide sequence of        SEQ ID NO: 31 or a probe or fragment prepared from the sequence,        under the stringent condition, and which has an activity to        support proliferation or survival of hematopoietic stem cell or        hematopoietic progenitor cell.-   (3) The DNA according to (2), the stringent condition is 6×SSC 5×    Denhardt, 0.5% SDS and 68° C. (SSC 3M NaCl, 0.3M sodium citrate, 50×    Denhardt 1% BSA 1% polyvinyl pyrrolidone, 1% Ficoll 400, or 6×SSC,    5× Denhardt, 0.5% SDS, 50% formamide and 42° C.-   (4) The DNA according to (1), which is a DNA as DEFINED in the    FOLLOWING (a) or (b):    -   (a) a DNA which comprises at least nucleotides 321 to 1074 of        SEQ ID NO: 33; or    -   (b) a DNA which is hybridizable with the nucleotide sequence of        SEQ ID NO: 33 or a prove prepared from the sequence, under the        stringent condition, and which has an activity to support        proliferation or survival of hematopoietic stem cell or        hematopoietic progenitor cell.-   (5) The DNA according to (4), the stringent condition is 6×SSC 5×    Denhardt 0.5% SDS and 68° C. (SSC 3M NaCl, 0.3M sodium citrate, 50×    Denhardt 1% BSA 1% polyvinyl pyrrolidone, 1% Ficoll 400, or 6×SSC,    5× Denhardt, 0.5% SDS, 50% Formamide and 42° C.-   (6) A expression vector which comprises a DNA of any one of (1)    to (5) or other polynucleotides of the invention in such a manner    that the DNA can be expressed.-   (7) A cell which is introduced (i.e., transformed or transfected)    with a DNA of any one of (1) to (5) or other polynucleotides of the    invention in such a manner that the DNA can be expressed.-   (8) A polypeptide (An isolated polypeptide) which is an expression    product of a DNA according to any one of (1) to (5) or other    polynucleotides of the invention, the polypeptide having an activity    to support proliferation or survival of hematopoietic stem cells or    hematopoietic progenitor cells, with a proviso that C-terminal amino    acid sequence dose not comprise the amino acid sequence of SEQ ID    NO: 14.-   (9) The polypeptide according to (8), which has an amino acid    sequence comprising at least amino acid residues 22 to 279 of SEQ ID    NO: 32, or an amino acid sequence including deletion, substitution    or insertion of one or several amino acids in the amino acid    sequence comprising at least amino acid residues 22 to 279 of SEQ ID    NO: 32.-   (10) The polypeptide according to (8), which has an amino acid    sequence comprising at least amino acid residues 22 to 272 of SEQ ID    NO: 34, or an amino acid sequence including deletion, substitution    or insertion of one or several amino acids in the amino acid    sequence comprising at least amino acid residues 22 to 272 of SEQ ID    NO: 34.-   (11) The polypeptide according to (8) or other polypeptides of the    invention, which is modified with one or more modifying agent    selected from the group consisting of polyethylene glycol (PEG),    dextran, poly(N-vinyl-pyrrolidone), polypropylene glycol homopoymer,    copolymer of polypropylene oxide/ethylene oxide, polyoxyethylated    polyol and polyvinyl alcohol.-   (12) Pharmaceutical composition having an effect to support    proliferation or survival of hematopoietic stem cells or    hematopoietic progenitor cells, which comprises the polypeptide as    defined in the following (A) (B) or (C): (A) a polypeptide which has    an amino acid sequence comprising at least amino acid residues 22 to    279 of SEQ ID NO: 32, or an amino acid sequence comprising at least    amino acid residues 22 to 272 of SEQ ID NO: 34; or    -   (B) a polypeptide which has an amino acid sequence including        deletion, substitution or insertion of one or several amino        acids in the amino acid sequence comprising at least amino acid        residues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence        comprising at least amino acid residues 22 to 272 of SEQ ID NO:        34, and which has an activity to support proliferation or        survival of hematopoietic stem cell or hematopoietic progenitor        cell, or    -   (C) any of the other polypeptides of the invention described        therein.-   (13) A monoclonal antibody which binds to the polypeptide of (9) to    (11).

Terms used in this specification are defined as follows.

A hematopoietic stem cell is defined as a cell having totipotency, thatis, a capacity to differentiate into all the cell lineages of thehematopoietic cells, and simultaneously having a potency of self-renewwith retaining the totipotency. Erythrocyte precursor cells hardlysurvive and proliferate in vitro culture circumstances and rapidlydisappear. If the survival and the proliferation of the erythrocyteprecursor cells are confirmed, continuous production of the erythrocyteprecursor cells seems to occur due to the survival and/or theproliferation of the more immature hematopoietic stem cells or thehematopoietic progenitor cells. Therefore, to assess the survival and/orproliferation of the human hematopoietic stem cells, to enumerate theerythrocyte precursor cells ((BFU-E, CFU-E, and CFU-Emix) in cultures isan appropriate way.

A hematopoietic progenitor cell is defined as a cell which candifferentiate a single cell lineage of the hematopoietic lineage or aplural cell lineages but cannot differentiate into all of the celllineages. A stromal cell is defined as a cell which can be co-culturedtogether with the hematopoietic stem cells in vitro to simulate in vivohematopoietic environment. Cells derived from any origin can be used aslong as the cells can be co-cultured with the hematopoietic cells invitro.

Polypeptides in accordance with the present invention have an activityto support proliferation or survival of hematopoietic stem cells orhematopoietic progenitor cells. The concrete embodiment of thepolypeptides in accordance with the present invention are an expressedproduct (hereafter sometimes referred to as a mouse “supporting factorfor the proliferation of stem cells”) of a gene named SCR-1 isolatedfrom a mouse stromal cell (hereafter sometimes referred to as “mouseSCR-1”) and an expressed product (hereafter sometimes referred to as ahuman “supporting factor for the proliferation of stem cells”) of ahuman orthologous gene thereof (hereafter sometimes referred to as“human SCR-1”). The term SCR-1 may be used herein to refer to thepolypeptide sequences set out in SEQ ID NO: 10, 13, 16, 32 and 34 whichare respectively encoded by the polnucleotide sequences set out in SEQID NOS: 9, 11, 12, 31 and 33.

Although an amino acid sequence of the expressed product of human SCR-1(SEQ ID NO: 34) has homology at 97.4% with the known polypeptide(WO98/49302) whose function has not been clear, the amino acid sequenceat the C-terminal region thereof differs, so that it is a novelpolypeptide. A part of the amino acid sequence in the above describedpolypeptide having unknown functions which is different from that in SEQID NO: 34 is shown in SEQ ID NO: 45. On the other hand, mouse SCR-1 hashomology at 84.6% with the above-described polypeptide.

The above described homologies are calculated as percentage of thenumber of same amino acids to the total number of amino acids using acomparison manually (266/273 and 237/280, respectively).

The supporting factor for the proliferation of stem cells, that is, thepolypeptides in accordance with the present invention can be produced bypreparing transformed cells by transducing mouse or human SCR-1 intoappropriate host cells and by expressing the DNAs in the transformedcells. When DNA including a nucleotide sequence shown in SEQ ID NO: 31is used as SCR-1, a mouse supporting factor for the proliferation ofstem cells is obtained. When DNA including a nucleotide sequence shownin SEQ ID NO: 33 is used as SCR-1, a human supporting factor for theproliferation of stem cells is obtained. The mouse supporting factor forthe proliferation of stem cells and the human supporting factor for theproliferation of stem cells comprise amino acid sequences represented bySEQ ID NO: 32 and SEQ ID NO: 34, respectively. These supporting factorsfor the proliferation of stem cells are precursors including signalpeptides, and are assumed to be processed to mature supporting factorsfor the proliferation of stem cells in mouse or human cells. As based onthe results of Signal P test which searches breakage sites of the signalpeptides in these amino acid sequences (Nielsen H., protein Engineering,10: 1-6, 1997; Nielsen H., Int. J. Neural Sys., 8: 581-599, 1997), thebreakage or cleavage sites seem to exist between the amino acid 21 andthe amino acid 22 in the amino acid sequences of SEQ ID NO: 32 and SEQID NO: 34.

The mouse mature supporting factor for the proliferation of stem cellscomprises the amino acid sequence represented by amino acids 22 to 279of SEQ ID NO: 32. The human mature supporting factor for theproliferation of stem cells comprises the amino acid sequencerepresented by amino acids 22 to 272 of SEQ ID NO: 34.

When supporting factors for the proliferation of stem cells areprepared, SCR-1 which is transferred into host cells may be DNA codingprecursor polypeptide or DNA coding mature polypeptide. An example ofthe DNA coding the mouse mature supporting factor for the proliferationof stem cells comprises the DNA comprising at least a nucleotidesequence consisting of nucleotide numbers 574 to 1347 of SEQ ID NO: 31.An example of the DNA coding the human mature supporting factor for theproliferation of stem cells comprises the DNA comprising at least anucleotide sequence consisting of nucleotide numbers 321 to 1074 of SEQID NO: 33.

DNA in accordance with the present invention may code the abovedescribed factors which have amino acid sequences includingsubstitution, deletion or insertion of one or several amino acids, aslong as the activity of the supporting factor for the proliferation ofstem cells to be coded is not lost. DNAs coding substantially identicalpolypeptides to this supporting factor for the proliferation of stemcells are obtained by modifying the nucleotide sequences so as toinclude substitution, deletion, insertion, addition, or inversion ofamino acid residues in a specific region using site-directedmutagenesis.

The DNAs including the above described mutation can be expressed inappropriate cells and the activity to support the hematopoietic stemcells of the expressed products can be examined, so that the DNAs codingthe polypeptide having functions which are substantially identical tothis supporting factor for the proliferation of stem cells are obtained.In addition, the DNAs coding substantially identically active protein asthis supporting factor for the proliferation of stem cells can beobtained by hybridization with DNAs including, for example, thenucleotide sequence as described in SEQ ID NO: 1 or SEQ ID NO: 3 fromthe cells having thereof, or probes prepared from these DNAs under thestringent condition; and by isolating the DNAs coding the proteinpossessing the activity to support the hematopoietic stem cells. Thestringent condition is, for example, one in which DNAs having homologyat not less than 70%, preferably at not less than 80%, are hybridizedeach other and DNAs having less homology than those are not hybridizedeach other. The above described stringent condition is 6×SSC, 5×Denhardt, 0.5% SDS, 68° C. (SSC; 3M NaCl, 0.3M sodium citrate) (50×Denhardt; 1% BSA, 1% polyvinyl pyrrolidone, 1% Ficoll 400) or 6×SSC, 5×Denhardt, 0.5% SDS, 50% Formamide, 42° C., or the like. Strategy ofhybridization is further defined by final wash conditions as set outherein.

Microorganisms such as Escherichia coli and yeast, culture cells derivedfrom animals or plants, and the like are used for host cells forexpressing SCR-1. Preferably, culture cells derived from mammals areused as the host cells. In the case that prokaryotic cells are used asthe host cells, the expression is preferably performed in a condition inwhich a signal peptide region is replaced with a leader sequencesuitable for the prokaryotic cells such as -lactamase (bla), alkalinephosphatase (phoA), and outer membrane protein A (ompA) and the like, orin a form in which a methionine residue is added to the N-terminal siteof the mature protein.

The supporting factor for the proliferation of stem cells obtained asabove may be added with sugar chains at any of positions 23, 36 and 137,alone, or a plurality of positions thereof in mouse SCR-1 The supportingfactor for the proliferation of stem cells obtained as above may beadded with sugar chains at any of positions 23, 36, 137 or 194, alone,or a plurality of positions thereof in human SCR-1.

For example, SCR-1 is integrated into a vector corresponding to the hostin a form capable of expression and the obtained recombinant vector istransferred into the host cells, so that the transfer of SCR-1 into thehost cells is completed.

Examples of the culture cells derived from mammals are CHO cells, 293cells, COS7 cells, and the like. Gene expression regulatory sequencesuch as a promoter to express SCR-1 may be originated from SCR-1 itself,or may be derived from other genes such as cytomegalovirus promoter andelongation factor 1 promoter and the like.

Examples of a vector for animal culture cells are plasmid vectors,retrovirus vectors, adenovirus vectors (Neering, S. J., Blood, 88: 1147,1996), herpes virus vectors (Dilloo, D., Blood, 89: 119, 1997), HIVvectors, and the like.

In order to transfer the recombinant vector into culture cells, theconventional methods which are usually employed for transformation ofculture cells such as calcium phosphate transfection, liposome method,DEAE dextran method, electroporation and microinjection method areemployed.

The polypeptides in accordance with the present invention also comprisepolypeptides having amino acid sequences in which one or several aminoacids are substituted, deleted or inserted in the amino acid sequencerepresented in SEQ ID NO: 32 or SEQ ID NO: 34 or other polynucleotidesof the invention, and having activity to support the hematopoietic stemcells in addition to the polypeptides having the amino acid sequencerepresented in SEQ ID NO: 32 or SEQ ID NO: 34 or other polynucleotidesof the invention. That is, even if a mouse and a human supporting factorfor the proliferation of stem cells is modified by substitution,deletion, insertion or the like, polypeptides holding essentialfunctions as a supporting factor for the proliferation of stem cells canbe considered to be substantially identical with the supporting factorfor the proliferation of stem cells. The above described “several”denotes ranging from two to 110, and preferably ranging from two to 55as a total number depending on the region of polypeptide in accordancewith the present invention.

These modified supporting factors for the proliferation of stem cellscan be obtained by treating DNA coding the supporting factors for theproliferation of stem cells or host cells including the above mentionedDNA with mutagens, or by mutating the above mentioned DNA so as tosubstitute, delete, or insert an amino acid at a specific site usingsite-directed mutagenesis. The residual of the activity to supporthematopoietic stem cells in the obtained mutant polypeptides isconfirmed by the examples described below. That is, after the culturedhematopoietic stem cells which express the mutant polypeptides aretransferred into irradiated mice, peripheral hematological cellularityafter the transfer may be observed over time.

Since the nucleotide sequences of the invention have been described, themodified supporting factor for the proliferation of stem cells can bealso obtained by isolating the corresponding DNAs from mouse or humancDNA or chromosome DNA libraries using PCR in which the oligonucleotidesprepared based on these nucleotide sequences are used as primers orusing hybridization in which the oligonucleotides prepared based onthese nucleotide sequences are used as probes.

In one aspect, the DNAs in accordance with the present invention wasisolated from cDNA library of AGM-s3-A9 cells which are a mouse stromalcell strain having the activity to support hematopoietic stem cellsusing SBH (Sequencing By Hybridization) method (Drmanac, S., Nat.Biotechnol., 16. 54, 1998; Drmanac, R., Methods. Enzymol., 303, 165,1999) as described below. The mouse stromal cell lines having theactivity to support hematopoietic stem cells can be obtained using themethod disclosed in WO99/03980 or from Cell Development Bank ofInstitute of Physical and Chemical Research (RIKEN) or ATCC.

An outline of SBH method will be described below. Probes including eightor nine nucleotides whose sequences are different from each other areprepared. When the nucleotide sequences corresponding to those of theprobe exist in targeted gene, the probes can hybridize with the gene.The hybridization can be easily detected with utilization of radioisotope or fluorescence conjugated probes. Each clone in the library ispicked up, and blotted on a membrane. Then, repeated hybridizations areperformed with the above described probes, so that one can identify thecombination of probes that hybridize to each clone. Since thecombination of probes that hybridize to each gene depend on thesequences of clones, identical genes have identical signaturehybridization patterns with the probes. That is, the same gene can beidentified as a one group (cluster) according to the signature of thehybridized probes. The number of clones derived from each gene in thelibrary can be determined by clustering and counting the members of theclusters based on the hybridization profiles of the probes. Thus,incidence of expression of each gene in the library can be determined.

Clustering analysis was performed for cDNA libraries derived fromsupportive and non-supportive stromal cell lines. Thus, incidences ofexpressed genes among cells were compared, so that the genesspecifically highly expressed in the supportive stromal cell lines wereselected. The incidences of these genes in each cell were furtherexamined by Northern blot analysis, so that genes which highly expressedin the cells having activity to support the hematopoietic stem cellswere obtained.

SCR-1 is one of the genes which was highly expressed with specificity inthe supporting cells obtained through the above described process. Afterclustering and analyzing using Northern blot analysis, the genecomprising nucleotide numbers 1032 to 1484 of SEQ ID NO: 31 wasidentified. The complete gene encoding SCR-1 was cloned from the cDNAlibrary derived from AGM-s3-A9 cells.

Further, in order to assess supporting ability for hematopoiesis ofSCR-1, a gene fragment including ORF (nucleotide numbers 511 to 1350 ofSEQ ID NO: 31) in SCR-1 gene was transferred into stromal cells(AGM-s3-A7 cell) which cannot support the hematopoietic stem cells usinga retrovirus vector, and assessed the change in the activity to supportthe hematopoietic stem cells of the stromal cells. Substantially, afterthe stromal cells which were not transferred with the gene and thosewhich were transferred with the gene were independently co-cultured withthe mouse hematopoietic stem cells, the hematopoietic cells weretransplanted into irradiated mice. Engraftment of the co-culturedhematopoietic cells in recipient mice were examined. As a result, themice transplanted with the hematopoietic stem cells which wereco-cultured with the AGM-s3-A7 cell line transferred with SCR-1 showedincreased chimerism after the transplantation compared with theAGM-s3-A7 cell line which were not transferred with SCR-1 gene. Thisresult shows that the AGM-s3-A7 stromal cells that express SCR-1 haveobtained supporting activity for the proliferation or survival of thehematopoietic stem cells or the hematopoietic progenitor cells. As aresult, it has become evident that SCR-1 has a function to add the abovedescribed activity to the stromal cells that originally do not possesthe activities for supporting proliferation or survival of hematopoieticstem cells or hematopoietic progenitor cells. Therefore, it was revealedthat SCR-1 has an activity to support the survival or the proliferationof the hematopoietic stem cell or the hematopoietic progenitor cell, orhas an activity to add an activity to support the hematopoietic stemcells to stromal cells.

The polypeptides in accordance with the present invention can be used asa medicine to proliferate or support human hematopoietic stem cell orhuman hematopoietic progenitor cell. This pharmaceutical composition canbe used for supporting proliferation or survival of human hematopoieticstem cells or human hematopoietic progenitor cells ex vivo. It is ofproblem for hematopoietic stem cell transplantation therapies such asperipheral blood stem cell transplantation and cord blood stem celltransplantation that sometimes sufficient amount of the hematopoieticstem cells cannot be collected and the transplantation may not beperformed. Even if enough stem cells could not be collected, asufficient amount of the hematopoietic stem cells could be obtained (andtransplanted) by amplification of the hematopoietic stem cells in vitrousing polypeptides of the invention. That is, hematopoietic stem cellscan be amplified without differentiation by culturing the hematopoieticstem cells in culture medium including polypeptides of the invention. Itmay be considered the hematopoietic stem cells are able to be amplifiedmore efficiently with addition of a variety of cytokines to the medium.

When hematopoietic stem cells or hematopoietic progenitor cells arecultured in the medium including the polypeptides in accordance with thepresent invention, the hematopoietic stem cells or the hematopoieticprogenitor cells that will be used may be one of these cell types aloneor may be both of the cell types. In addition, the cells should includeat least the hematopoietic stem cells or the hematopoietic progenitorcells, and may include other hematopoietic cells. Further, polypeptidesof the invention can be used for hematopoietic stem or progenitorexpansion of purified hematopoietic stem cell fraction or progenitorcell fractions from the cell populations that contain the hematopoieticstem cells or progenitor cells.

Examples of sources of hematopoietic stem cells and hematopoieticprogenitor cells in the methods in accordance with the present inventionare fetal liver, bone marrow, fetal bone marrow, peripheral blood,peripheral blood from persons from whom stem cells are mobilized bycytokines and/or dosing of antitumor drugs, cord blood, and the like ofmammals such as human and mouse and the like. Any sources may be used aslong as the tissue includes the hematopoietic stem cells.

A culture method using petri dishes and flasks for culture may beemployed to culture the hematopoietic stem cells or the hematopoieticprogenitor cells. The cultivation of the hematopoietic stem cells and/orprogenitor cells may be improved by mechanically controlling mediumcomposition, pH, and the like, and employing a bioreactor capable ofhigh density cultivation (Schwartz, Proc. Natl. Acad. Sci. U.S.A., 88:6760, 1991; Koller, M. R., Bio/Technology, 11: 358, 1993; Koller, M. R.,Blood, 82: 378, 1993; Palsson, B. O., Bio/Technology, 11: 368, 1993).

Since SCR-1 can increase activities of stromal cells to support thehematopoietic stem cells under the conditions of co-culture of stromalcells and hematopoietic cells, the hematopoietic stem cells and/orprogenitor cells can be efficiently expanded when whole bone marrowcells are cultured in the presence of SCR-1. This type of co-culture ofthe stromal cells and the hematopoietic cells can be performed simplyafter the collection of the bone marrow cells without complicated cellseparation. Furthermore, one can perform co-culture with separatecomponents such as hematopoietic stem cells, progenitor cells andstromal cells from collected bone marrow cells and combine thehematopoietic cells and stromal cells from different individuals.Furthermore, one can grow stromal cells and establish stromal cellculture prior to co-culture with the hematopoietic stem cells for thehematopoietic stem cells or progenitor cell expansion. At this time, onecan utilize cell stimulating factors to promote growth and survival ofstromal cells to establish stromal cell culture. Examples of cellstimulating factors includes growth factors which are typically acytokine such as SCF (stem cell factor), IL-3 (interleukin-3), GM-CSF(granulocyte/macrophage colony-stimulating factor), IL6 (interleukin-6),TPO (thrombopoietin), G-CSF (granulocyte colony-stimulating factor),TGF-b (transforming growth factor-b), MIP-1a (Davatelis, G., J. Exp.Med. 167: 1939, 1988); differentiation and proliferation control factorssuch as hematopoietic hormones such as EPO (erythropoietin), chemokine,Wnt gene product, and notch ligand; and development control factors.

In addition, the proliferation and the survival of hematopoietic stemcells or hematopoietic progenitor cells can be retained by culturing thehematopoietic stem and/or progenitor cells with recombinant SCR-1 aloneor combination with the cell stimulating factors without stromal cells.Examples of the cell stimulating factors used in this case are abovedescribed cell stimulating factors and the like.

Medium used for culture is not specially restricted as long as theproliferation or the survival of the hematopoietic stem cells or thehematopoietic progenitor cells is not perturbed. Preferable media are,for example, MEM-α medium (GIBCO BRL), SF-02 medium (Sanko Junyaku),Opti-MEM medium (GIBCO BRL), IMDM medium (GIBCO BRL), and PRMI1640medium (GIBCO BRL). A culture temperature is usually ranging from 25 to39° C., and preferably ranging from 33 to 39° C. Examples of additivesto the medium are fetal bovine serum, human serum, horse serum, insulin,transferrin, lactoferrin, ethanolamine, sodium selenite,monothiolglycerol, 2-mercaptoethanol, bovine serum albumin, sodiumpyruvate, polyethylene glycol, a variety of vitamins, and a variety ofamino acids. A concentration of CO₂ is usually ranging from four to sixpercent, and preferably five percent.

Since hematopoietic stem cells can differentiate into all hematopoieticcell lineages, hematopoietic stem cells can be manipulated to bedifferentiated into a specific cell type in vitro, and then the specificcells can be transplanted. For example, when erythrocytes are necessary,after cultivation and expansion of the patient's stem cells, hemopoieticcells whose main component is the erythrocyte can be artificiallyproduced using an erythrocyte differentiation induction or promotingfactors such as EPO.

The hematopoietic stem cells or the hematopoietic progenitor cellscultured using the polypeptides in accordance with the present inventioncan replace as a graft for the conventional bone marrow transplantationor cord blood transplantation. Transplantation of the hematopoietic stemcells is superior to the conventional hematopoietic cell transplantationtherapy, since the graft can take semipermanently.

The transplantation of the hematopoietic stem cells can be employed astherapy for a variety of diseases in addition to as combination therapyfor total body X-ray irradiation therapy or advanced chemotherapy forleukemia. For example, when therapy accompanied with myelosuppression asan adverse reaction such as chemotherapy, radiation therapy, and thelike is performed for the patient with solid cancer, hematologicaldisorder, hematological failure can be early improved as follows. Thebone marrow is collected before the therapy and the hematopoietic stemcells or the hematopoietic progenitor cells are allowed to expand invitro. Then, the expanded cells are infused to the patient after thetherapy, so that the patient can get benefit of early recovery andstronger chemotherapy than the conventional one can be performed toimprove the therapeutic effect of the chemotherapy. In addition, thehematopoietic stem cells or the hematopoietic progenitor cells obtainedaccording to the present invention are differentiated into a variety ofhematopoietic cells. The transplantation of these cells into a patientwith hypoplasia of a given hematopoietic cells can improve the patient'sdeficient status. In addition, this therapy can improve dyshemopoieticanemia to develop anemia such as aplastic anemia caused by bone marrowhypoplasia. Furthermore, examples of diseases in which thetransplantation of the hematopoietic stem cells according to the presentinvention is effective are immunodeficiency syndrome such as chronicgranulomatous disease, duplicated immunodeficiency syndrome,agammaglobulinemia, Wiskott-Aldrich syndrome, acquired immunodeficiencysyndrome (AIDS), and the like, thalassemia, hemolytic anemia due toenzyme defect, congenital anemia such as sicklemia, Gaucher's disease,lysosomal storage disease such as mucopolysaccharidosis, adrenal whitematter degeneration, a variety of cancers and tumors, and the like.

Transplantation of hematopoietic stem cells may be performed in the samemanner as conventional bone marrow transplantation or cord bloodtransplantation other than the differences of the cells used.

The hematopoietic stem cells which may be used for the above describedhematopoietic stem cell transplantation are derived from not only bonemarrow but also the above described fetal liver, fetal bone marrow,peripheral blood, peripheral blood with stem cells induced by cytokinesand/or dosing of antitumor drugs, cord blood, and the like.

The graft may be a composition including buffer solution and the like inaddition to the hematopoietic stem cells and the hematopoieticprogenitor cells produced by the method according to the presentinvention.

The hematopoietic stem cells or the hematopoietic progenitor cellsproduced according to the present invention may be used for ex vivo genetherapy. Since the incidence of recombination of target genes to thechromosome is low due to dormancy of the stem cells, differentiation ofstem cells during the culture period, and the like, gene therapy to thehematopoietic stem cells has been hard to established. However, thepresent invention can amplify stem cells without differentiation, sothat efficacy of gene transfer is expected to be remarkably improved. Ingene therapy, a foreign gene (a gene for therapy) is transferred intothe hematopoietic stem cells or the hematopoietic progenitor cells, andthen the obtained gene-transferred cells are used. The foreign gene tobe transferred is appropriately selected according to disease. Examplesof diseases in which the target cells of gene therapy is thehematopoietic cells include immunodeficiency syndrome such as chronicgranulomatous disease, duplicated immunodeficiency syndrome,agammaglobulinemia, Wiskott-Aldrich syndrome, acquired immunodeficiencysyndrome (AIDS), and the like, thalassemia, hemolytic anemia due toenzyme defect, congenital anemia such as sicklemia, Gaucher's disease,lysosomal storage disease such as mucopolysaccharidosis, adrenal whitematter degeneration, a variety of cancers and tumors, and the like.

Usual method used for transfer of a gene into animal cells is employedfor the transfer of the gene for the therapy into hematopoietic stemcells or hematopoietic progenitor cells. Examples are a method using avector for animal cells derived from virus utilized for gene therapysuch as retrovirus vector such as Moloney mouse leukemia virus,adenovirus vector, adeno-associated virus (AAV) vector, herpes simplexvirus vector, and HIV vector (with respect to a vector for gene therapy,see Verma, I. M., Nature, 389: 239, 1997); calcium phosphatetransfection, DEAE-dextran transfection, electroporation, liposomemethod, lipofection method, microinjection method, and the like. Amongthem, methods using retrovirus vector, adeno-associated virus vector, orHIV vector are preferable, since expression of a gene is permanentlyexpected due to insertion into the chromosome DNA of a target cell.

For example, adeno-associated virus (AAV) vector can be prepared asfollows. First, a vector plasmid inserted a gene for therapy into ITR(inverted terminal repeat) at both ends of wild-type adeno-associatedvirus DNA and a helper plasmid for supplementing virus protein aretransfected into 293 cell strain. Next, adenovirus as helper virus isinfected, so that virus particles including the AAV vector are produced.Alternatively, instead of adenovirus, a plasmid which expressesadenovirus gene coding helper function may be transfected. The obtainedvirus particles are infected to the hematopoietic stem cells or thehematopoietic progenitor cells. Preferably, appropriate promoter andenhancer are inserted into upstream region of the target gene in thevector DNA, so that the expression of the gene is regulated. When markergene such as a drug resistant gene is used in addition to the gene fortherapy, cells transferred with the gene for therapy are easilyselected. The gene for therapy may be sense gene or antisense gene.

A composition for gene therapy may include buffer solution and a novelactive ingredient and the like in addition to the hematopoietic stemcells or the hematopoietic progenitor cells by the method according tothe present invention.

A vector for gene therapy can be produced by transferring SCR-1 inexpression vector using a usual method. This vector for gene therapy isuseful to treat diseases which need survival and proliferation of thehuman hematopoietic stem cells. That is, a vector producing SCR-1 istransferred into the hematopoietic stem cells and the cells are culturedin vitro, so that the hematopoietic stem cells or the hematopoieticprogenitor cells can proliferate dominatingly. The hematopoietic stemcells can proliferate in vivo caused by returning these hematopoieticstem cells thus treated. The hematopoietic stem cells can significantlyproliferate in vivo by introducing this vector for gene therapy into thebody. Alternatively, the bone marrow cells derived from a patient arecultured and transferred with this vector for gene therapy, so that thehematopoietic stem cells or the hematopoietic progenitor cells can beproliferated in culture system. Alternatively, this vector for genetherapy is transferred into stromal cell derived from bone marrow andcultivated and mesenchaymal stem cell, so that the activity to supporthematopoietic stem cells can be added or increased.

As shown in Examples, since it is possible that the stromal cellswithout the activity to support the hematopoietic stem cells can bemodified to include this activity using SCR-1, stromal cells derivedfrom human or mouse can have the activity to support the hematopoieticstem cells by gene transferring SCR-1. The stromal cells expressingSCR-1 and hematopoietic stem cells or hematopoietic progenitor cells areco-cultured, so that the hematopoietic stem cells or the hematopoieticprogenitor cells can exist and proliferate so as to be useful for avariety treatment.

Since the hematopoietic stem cells or the hematopoietic progenitor cellscan survive and proliferate by expression of SCR-1 in the stromal cell,an activity to support the hematopoietic stem cells of the stromal cellscan be assessed using the expression of SCR-1 as an index. Theexpression of SCR-1 in the stromal cells can be confirmed using antibodyto SCR-1. PCR primers can be prepared from genes included in SEQ ID NO:31, SEQ ID NO: 33 or other polynucleotides of the invention and RNA isprepared from the stromal cells of interest, and RT-PCR is performed, sothat the expression of SCR-1 can be confirmed. Antibody to SCR-1 will bedescribed below.

The pharmaceutical composition in accordance with the present inventioncan be administered to human. The pharmaceutical composition having anactivity to proliferate or to support the human hematopoietic stem cellsor the hematopoietic progenitor cells can be produced by mixingmedically acceptable diluent, stabilizer, carrier, and/or otheradditives with the polypeptides in accordance with the presentinvention. At this time, in order to increase the stability of theprotein in vivo the polypeptides in accordance with the presentinvention may be modified by a modifying agent. Examples of themodifying agent are polyethylene glycol (PEG), dextran,poly(N-vinyl-pyrrolidone), polypropylene glycol homopolymer,polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol,polyvinyl alcohol, and the like. Examples of modification of proteinwith PEG are a method in which activated ester derivatives of PEG isreacted with the protein, a method in which aldehyde derivatives at endportion of PEG is reacted with protein under the presence of a reducingagent, and the like. Japanese Unexamined Patent Application No.10-510980 discloses modification of such protein in detail.

When the pharmaceutical composition in accordance with the presentinvention is administered to human, recovery from hematologicalsuppression due to an adverse drug reaction of carcinostatics; earlyrecovery of hematopoietic cells at bone marrow transplantation,peripheral blood stem cell transplantation, or cord bloodtransplantation; and recovery of hematopoietic function at pancytopeniasuch as aplastic anemia (AA) and myelodysplastic syndrome (MDS) areexpected.

The antibody in accordance with the present invention reactsspecifically to the above described polypeptides in accordance with thepresent invention. This antibody may be monoclonal antibodies orpolyclonal antibodies as long as they react specifically to the abovedescribed polypeptides.

The antibody in accordance with the present invention can be preparedaccording to usual methods. For example, the antibody can be preparedeither in vivo method in which animals are additionally immunized byantigen together with adjuvant once or several times at an interval ofseveral weeks or in vitro method in which immune cells are isolated andsensitized in an appropriate culture system. Examples of immune cellswhich can produce the antibody in accordance with the present inventionare splenic cells, tonsillar cells, lymph gland cells, and the like.

The whole polypeptide according to the present invention is notnecessarily used as an antigen. A part of a polypeptide of the inventionmay be used as an antigen. When the antigen is a short peptide,particularly, a peptide made of about 20 amino acid residues, it may beused by binding it to a carrier protein having high antigenicity such askeyhole lympet hemocyanin or bovine serum albumin using chemicalmodification and the like. Alternatively, the antigen may be used bycovalently binding it to a peptide having branching skeleton such aslysine core MAP peptide instead of the carrier protein (Posnett-et al.,J. Biol. Chem., 263, 1719-1725, 1988; Lu et al., Mol. Immunol., 28,623-630, 1991; Briand et al., J. Immunol. Methods, 156, 255-265, 1992).

Examples of adjuvants are Freund's complete adjuvant, Freund'sincomplete adjuvant, aluminum hydroxide gel, and the like. Animals giventhe antigen are, for example, mouse, rat, rabbit, sheep, goat, chicken,bovine, horse, guinea pig, hamster, and the like. The blood is collectedfrom these animals and the serum is separated. Then, immunoglobulin ispurified from the serum using an ammonium sulfate precipitation method,anion exchange chromatography, protein A chromatography, or protein Gchromatography to obtain polyclonal antibodies.

With respect to chicken, antibodies can be purified from an egg.Monoclonal antibodies can be purified and prepared from supernatant ofculture of hybridoma cells or ascites from animals which receivedintrapertoneal administration of hybridoma cells. Hybridomas are made byfusion of the immune cells sensitized in vitro, or immune cells from theabove described animals with parent cells capable of cultivation.Examples of parent cells are X63, NS-1, P3U1, X63.653, SP2/O, Y3,SKO-007, GM1500, UC729-6, HM2.0, NP4-1 cell strains, and the like.Preparation may be performed by cultivating the immortalizedantibody-forming cells obtained by sensitization in vitro, or infectionof a proper virus such as EB virus to the immune cells of the abovedescribed animals.

In addition to these cell engineering methods, antibodies can beobtained using gene engineering methods. For example, the antibody geneobtained from the in vitro sensitized cells or immune cells derived fromthe above described animals is amplified by PCR (polymerase chainreaction) and isolated, and the amplified genes are transferred intomicroorganisms such as E. coli to produce the antibodies. Alternatively,the antibodies may be expressed on surfaces of phages as fused protein.Antibodies of the invention are also addressed herein, infra.

SCR-1 can be measured in vivo using antibodies in accordance with thepresent invention. Thus, the relationship between SCR-1 and pathologicalstatus of a variety of diseases can be clarified. Moreover, theantibodies can be used for diagnosis and treatment of diseases, andefficient affinity purification of SCR-1.

The present invention provides polypeptides having an activity tosupport survival or proliferation of hematopoietic stem cells orhematopoietic progenitor cells by effecting or acting thereon, or anactivity to give an activity to support the hematopoietic stem cells tostromal cells by effecting thereon, and also provides DNA codingthereof. The polypeptides in accordance with the present invention canefficiently maintain the proliferation or the survival of thehematopoietic stem cells or the hematopoietic progenitor cells.

In addition, the polypeptides in accordance with the present inventioncan be used as a medicine to proliferate or to support humanhematopoietic stem cells or human hematopoietic progenitor cells.

Alternatively, the invention is described as set out below.

5.2 Nucleic Acids and Polypeptides of the Invention

Nucleotide and amino acid sequences of the invention are set forth asSEQ ID NO: 1-24, and 31-34. Fragments of the proteins of the presentinvention which are capable of exhibiting biological activity are alsoencompassed by the present invention. Fragments of the protein may be inlinear form or they may be cyclized using known methods, for example, asdescribed in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992)and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253(1992), both of which are incorporated herein by reference. Suchfragments may be fused to carrier molecules such as immunoglobulins formany purposes, including increasing the valency of protein bindingsites. For example, fragments of the protein may be fused through“linker” sequences to the Fc portion of an immunoglobulin. For abivalent form of the protein, such a fusion could be to the Fc portionof an IgG molecule. Other immunoglobulin isotypes may also be used togenerate such fusions. For example, a protein-IgM fusion would generatea decavalent form of the protein of the invention.

The present invention also provides both full-length and mature forms(for example, without a signal sequence or precursor sequence) of thedisclosed proteins. The protein coding sequence is identified in thesequence listing by translation of the disclosed nucleotide sequences.The mature form of such protein may be obtained by expression of afull-length polynucleotide in a suitable mammalian cell or other hostcell. The sequence of the mature form of the protein is alsodeterminable from the amino acid sequence of the full-length form. Whereproteins of the present invention are membrane bound, soluble forms ofthe proteins are also provided. In such forms, part or all of theregions causing the proteins to be membrane bound are deleted so thatthe proteins are fully secreted from the cell in which it is expressed.

The polynucleotides of the invention include naturally occurring orwholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA,e.g., mRNA. SEQ ID NO: 1-9, 11, 12, 31 or 33 may include the entirecoding region of the cDNA or may represent a portion of the codingregion of the cDNA. Further 5′ and 3′ sequence can be obtained usingmethods known in the art. For example, full length cDNA or genomic DNAthat corresponds to any of the polynucleotides of the SEQ ID NO: 1-9,11, 12, 31 or 33 can be obtained by screening appropriate cDNA orgenomic DNA libraries under suitable hybridization conditions using anyof the polynucleotides of the SEQ ID NO: 1-9, 11, 12, 31 or 33 or aportion thereof as a probe. Alternatively, the polynucleotides of theSEQ ID NO: 1-9, 11, 12, 31 or 33 may be used as the basis for suitableprimer(s) that allow identification and/or amplification of genes inappropriate genomic DNA or cDNA libraries.

The nucleic acid sequences of the invention can be assembled ESTs andsequences (including cDNA and genomic sequences) obtained from one ormore public databases, such as dbEST, gbpri, and UniGene. The sequencesfalling within the scope of the present invention are not limited tothese specific sequences, but also include allelic and speciesvariations thereof. Allelic and species variations can be routinelydetermined by comparing the sequence provided in SEQ ID NO: 8-9, 11-12,31 or 33 a representative fragment thereof, or a nucleotide sequence atleast 90% identical, preferably 99.9% identical, to SEQ ID NO: 8-9,11-12, 31 or 33 with a sequence from another isolate of the samespecies. Furthermore, to accommodate codon variability, the inventionincludes nucleic acid molecules coding for the same amino acid sequencesas do the specific ORFs disclosed herein. In other words, in the codingregion of an ORF, substitution of one codon for another which encodesthe same amino acid is expressly contemplated.

The nucleic acids of the present invention, designated as SEQ ID NO. 8and 9 were assembled using an EST sequence as a seed. The EST sequencecan be extended into a full-length nucleic acid sequence by programs oralgorithms known in the art. Preferably, a recursive algorithm is usedto extend the seed EST into an extended assemblage, by pullingadditional sequences from different databases (e.g., Hyseq's databasecontaining EST sequences, dbEST version 114, gb pri 114, and UniGeneversion 101) that belong to this assemblage. The algorithm terminateswhen there was no additional sequences from the databases that willextend the assemblage. Further, the inclusion of component sequencesinto the assemblage is preferably based on a BLASTN hit to the extendingassemblage with BLAST score greater than 300 and percent identitygreater than 95%. BLAST, which stands for Basic Local Alignment SearchTool, is used to search for local sequence alignments (Altschul, S. F.,J. Mol. Evol. 36: 290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21: 403-10 (1990)). BLAST produces alignments of both nucleotideand amino acid sequences to determine sequence similarity. Because ofthe local nature of the alignments, BLAST is especially useful indetermining exact matches.

The EST sequences (SEQ ID NO. 1-7) can provide identifying sequenceinformation, representative fragment or segment information, or novelsegment information for the fill-length gene.

The nearest neighbor result for the nucleic acids of the presentinvention, including SEQ ID NO. 9, can be obtained by searching adatabase using an algorithm or a program. Preferably, a FASTA version 3search against Genpept, using Fastxy algorithm. The nearest neighborresult shows the closest homologue for each assemblage from Genpept (andcontains the translated amino acid sequences for which the assemblageencodes).

The present invention also provides genes corresponding to the cDNAsequences disclosed herein. The corresponding genes can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include the preparation of probes or primers fromthe disclosed sequence information for identification and/oramplification of genes in appropriate genomic libraries or other sourcesof genomic materials.

Species homologs (or orthologs) of the disclosed polynucleotides andproteins are also provided by the present invention. Species homologsmay be isolated and identified by making suitable probes or primers fromthe sequences provided herein and screening a suitable nucleic acidsource from the desired species.

The invention also encompasses allelic variants of the disclosedpolynucleotides or proteins; that is, naturally-occurring alternativeforms of the isolated polynucleotide which also encode proteins whichare identical, homologous or related to that encoded by thepolynucleotides.

5.3 Nucleic Acids of the Invention

The isolated polynucleotides of the invention include, but are notlimited to, a polynucleotide encoding a polypeptide comprising SEQ IDNO: 10, 13-24, 32 and 34, or the mature protein portion thereof.Preferred nucleic acid sequences are set forth as SEQ ID NO: 9, 11, 12,31 or 33.

The isolated polynucleotides of the invention further include, but arenot limited to a polynucleotide comprising any of the nucleotidesequence of the SEQ ID NO: 1-9, 11, 12, 31 or 33; a polynucleotidecomprising the full length protein coding sequence of thepolynucleotides of the SEQ ID NO: 1-9, 11, 12, 31 or 33; and apolynucleotide comprising the nucleotide sequence encoding the matureprotein coding sequence of the polynucleotides of the SEQ ID NO: 1-9,11, 12, 31 or 33. The polynucleotides of the present invention alsoinclude, but are not limited to, a polynucleotide that preferably hasstem cell growth factor activity and that hybridizes under stringentconditions (a) to the complement of any of the nucleotides sequences ofthe SEQ ID NO: 1-9, 11, 12, 31 or 33 (b) to a polynucleotide encodingthe polypeptide of SEQ ID NO: 10, 13-24, 32 or 34, a polynucleotidewhich is an allelic variant of any polynucleotide recited above; apolynucleotide which encodes a species homolog of any of the proteinsrecited above; or a polynucleotide that encodes a polypeptide comprisinga specific domain or truncation of the polypeptide of SEQ ID NO: 10,13-24, 32 or 34. Domains of interest may depend on the nature of theencoded polypeptide; e.g., domains in receptor-like polypeptides includeligand-binding, extracellular, transmembrane, or cytoplasmic domains, orcombinations thereof; domains in immunoglobulin-like proteins includethe variable immunoglobulin-like domains; domains in enzyme-likepolypeptides include catalytic and substrate binding domains; anddomains in ligand polypeptides include receptor-binding domains.

Polynucleotides encoding preferred polypeptide truncations of theinvention can be used to generate polynucleotides encoding chimeric orfusion proteins comprising one or more domains of the invention andheterologous protein sequences.

The polynucleotides of the invention additionally include the complementof any of the polynucleotides recited above.

The polynucleotides of the invention also provide polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides recited above. Polynucleotides according to theinvention can have, e.g., at least about 65%, at least about 70%, atleast about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typicallyat least about 85%, 86%, 87%, 88%, 89%, more typically at least about90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%,96%, 97%, 98%, 99% sequence identity to a polynucleotide recited above.The invention also provides the complement of such polynucleotides. Thepolynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) orRNA. Methods and algorithms for obtaining such polynucleotides are wellknown to those of skill in the art and can include, for example, methodsfor determining hybridization conditions which can routinely isolatepolynucleotides of the desired sequence identities.

A polynucleotide according to the invention can be joined to any of avariety of other nucleotide sequences by well-established recombinantDNA techniques (see Sambrook J et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotidesequences for joining to polynucleotides include an assortment ofvectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids,and the like, that are well known in the art. Accordingly, the inventionalso provides a vector including a polynucleotide of the invention and ahost cell containing the polynucleotide. In general, the vector containsan origin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell. Vectors according to the invention include expression vectors,replication vectors, probe generation vectors, and sequencing vectors. Ahost cell according to the invention can be a prokaryotic or eukaryoticcell and can be a unicellular organism or part of a multicellularorganism.

The sequences falling within the scope of the present invention are notlimited to the specific sequences herein described, but also includeallelic variations thereof. Allelic variations can be routinelydetermined by comparing the nucleotide sequences provided in the SEQ IDNO: 1-9, 11, 12, 31 or 33, a representative fragment thereof, or anucleotide sequence at least 99.9% identical to any of the nucleotidesequences of the SEQ ID NO: 1-9, 11, 12, 31 or 33 with a sequence fromanother isolate of the same species. To accommodate codon variability,the invention includes nucleic acid molecules coding for the same aminoacid sequences as do the specific ORFs disclosed herein. In other words,in the coding region of an ORF, substitution of one codon for anotherwhich encodes the same amino acid is expressly contemplated. Anyspecific sequence disclosed herein can be readily screened for errors byresequencing a particular fragment, such as an ORF, in both directions(i.e., sequence both strands).

The present invention further provides recombinant constructs comprisinga nucleic acid having any of the nucleotide sequences of the SEQ ID NO:1-9, 11, 12, 31 or 33 or a fragment thereof or any other polynucleotidesof the invention. In one embodiment, the recombinant constructs of thepresent invention comprise a vector, such as a plasmid or viral vector,into which a nucleic acid having any of the nucleotide sequences of theSEQ ID NO: 1-9, 11, 12, 31 or 33 or a fragment thereof is inserted, in aforward or reverse orientation. In the case of a vector comprising oneof the ORFs of the present invention, the vector may further compriseregulatory sequences, including for example, a promoter, operably linkedto the ORF. For vectors comprising the EMFs and UMFs of the presentinvention, the vector may further comprise a marker sequence orheterologous ORF operably linked to the EMF or UMF. Large numbers ofsuitable vectors and promoters are known to those of skill in the artand are commercially available for generating the recombinant constructsof the present invention. The following vectors are provided by way ofexample. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein“operably linked” means that the isolated polynucleotide of theinvention and an expression control sequence are situated within avector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art. Generally, recombinant expressionvectors will include origins of replication and selectable markerspermitting transformation of the host cell, e.g., the ampicillinresistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoterderived from a highly-expressed gene to direct transcription of adownstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including an aminoterminal identification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed. Followingtransformation of a suitable host strain and growth of the host strainto an appropriate cell density, the selected promoter is induced orderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

Included within the scope of the nucleic acid sequences of the inventionare nucleic acid sequence fragments that hybridize under stringentconditions to any of the nucleotide sequences of the SEQ ID NO: 1-9, 11,12, 31 or 33 or complements thereof, which fragment is greater thanabout 10 bp, preferably 20 to 50 bp, and even greater than 100 bp,greater than 300 bp, or greater than 500 bp. Fragments of, e.g. 15, 16,or 20 bp or more that are selective for (i.e. specifically hybridize toany one of the polynucleotides of the invention) are contemplated.Probes capable of specifically hybridizing to a polynucleotide candifferentiate polynucleotide sequences of the invention from otherpolynucleotide sequences in the same family of genes or candifferentiate human genes from genes of other species, and arepreferably based on unique nucleotide sequences.

In accordance with the invention, polynucleotide sequences comprisingthe mature protein coding sequences corresponding to the SEQ ID NO: 10,13-24, 32 or 34 or functional equivalents thereof, may be used togenerate recombinant DNA molecules that direct the expression of thatnucleic acid, or a functional equivalent thereof, in appropriate hostcells. Also included are the cDNA inserts of any of the clonesidentified herein.

The nucleic acid sequences of the invention are further directed tosequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide changes into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. Nucleic acids encoding the amino acid sequence variantsare preferably constructed by mutating the polynucleotide to encode anamino acid sequence that does not occur in nature. These nucleic acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite. Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino and/or carboxyl-terminalfusions ranging in length from one to one hundred or more residues, aswell as intrasequence insertions of single or multiple amino acidresidues. Intrasequence insertions may range generally from about 1 to10 amino residues, preferably from 1 to 5 residues. Examples of terminalinsertions include the heterologous signal sequences necessary forsecretion or for intracellular targeting in different host cells andsequences such as FLAG or poly-histidine sequences useful for purifyingthe expressed protein.

In a preferred method, polynucleotides encoding the novel amino acidsequences are changed via site-directed mutagenesis. This method usesoligonucleotide sequences to alter a polynucleotide to encode thedesired amino acid variant, as well as a sufficient adjacent nucleotideson both sides of the changed amino acid to form a stable duplex oneither side of the site of being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may alsobe used to create amino acid sequence variants of the novel nucleicacids. When small amounts of template DNA are used as starting material,primer(s) that differs slightly in sequence from the correspondingregion in the template DNA can generate the desired amino acid variant.PCR amplification results in a population of product DNA fragments thatdiffer from the polynucleotide template encoding the polypeptide at theposition specified by the primer. The product DNA fragments replace thecorresponding region in the plasmid and this gives a polynucleotideencoding the desired amino acid variant.

A further technique for generating amino acid variants is the cassettemutagenesis technique described in Wells et al., Gene 34:315 (1985); andother mutagenesis techniques well known in the art, such as, forexample, the techniques in Sambrook et al., supra, and Current Protocolsin Molecular Biology, Ausubel et al. Due to the inherent degeneracy ofthe genetic code, other DNA sequences which encode substantially thesame or a functionally equivalent amino acid sequence may be used in thepractice of the invention for the cloning and expression of these novelnucleic acids. Such DNA sequences include those which are capable ofhybridizing to the appropriate novel nucleic acid sequence understringent conditions.

Polynucleotides of the invention can also be used to induce immuneresponses. For example, as described in Fan et al., Nat. Biotech.17:870-872 (1999), incorporated herein by reference, nucleic acidsequences encoding a polypeptide may be used to generate antibodiesagainst the encoded polypeptide following topical administration ofnaked plasmid DNA or following injection, and preferably intramuscularinjection of the DNA. The nucleic acid sequences are preferably insertedin a recombinant expression vector and may be in the form of naked DNA.

5.3.1 Antisense Nucleic Acids

Another aspect of the invention pertains to isolated antisense nucleicacid molecules that can hybridize to, or are complementary to, thenucleic acid molecule comprising the stem cell growth factor-likenucleotide sequence, or fragments, analogs or derivatives thereof. An“antisense” nucleic acid comprises a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein (e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence). In specific aspects, antisensenucleic acid molecules are provided that comprise a sequencecomplementary to at least about 10, 25, 50, 100, 250 or 500 nucleotidesor an entire stem cell growth factor-like coding strand, or to only aportion thereof. Nucleic acid molecules encoding fragments, homologs,derivatives, and analogs of a stem cell growth factor-like or antisensenucleic acids complementary to a stem cell growth factor-like nucleicacid sequence of are additionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encoding astem cell growth factor-like protein. The term “coding region” refers tothe region of the nucleotide sequence comprising codons which aretranslated into amino acid residues. In another embodiment, theantisense nucleic acid molecule is antisense to a “conceding region” ofthe coding strand of a nucleotide sequence encoding the stem cell growthfactor-like protein. The term “conceding region” refers to 5′ and 3′sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding the stem cell growthfactor-like protein disclosed herein, antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crick orHoogsteen base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of stem cell growthfactor-like mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region of stemcell growth factor-like mRNA. For example, the antisense oligonucleotidecan be complementary to the region surrounding the translation startsite of stem cell growth factor-like mRNA. An antisense oligonucleotidecan be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis or enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids (e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used).

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaniinomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following section).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a stem cellgrowth factor-like protein thereby inhibit expression of the protein(e.g., by inhibiting transcription and translation). The hybridizationcan be by conventional nucleotide complementarity to form a stableduplex, or, for example, in the case of an antisense nucleic acidmolecule that binds to DNA duplexes, through specific interactions inthe major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface (e.g., bylinking the antisense nucleic acid molecules to peptides or antibodiesthat bind to cell surface receptors or antigens). The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient nucleic acid molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an alpha-anomeric nucleic acid molecule. An alpha-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual alpha-units, thestrands run parallel to each other. See, e.g., Gaultier, et al., 1987.Nucl. Acids Res. 15, 6625-6641. The antisense nucleic acid molecule canalso comprise a 2′-o-methylribonucleotide (see, e.g., Inoue, et al.1987. Nucl. Acids Res. 15, 6131-6148) or a chimeric RNA-DNA analogue(see, e.g., Inoue, et al., 1987. FEBS Lett. 215, 327-330).

5.3.2 Ribozymes and PNA Moieties

Nucleic acid modifications include, by way of non-limiting example,modified bases, and nucleic acids whose sugar phosphate backbones aremodified or derivatized. These modifications are carried out at least inpart to enhance the chemical stability of the modified nucleic acid,such that they can be used, for example, as antisense binding nucleicacids in therapeutic applications in a subject.

In one embodiment, an antisense nucleic acid of the invention is aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes as described in Haselhoff andGerlach 1988. Nature 334, 585-591) can be used to catalytically cleavestem cell growth factor-like mRNA transcripts to thereby inhibittranslation of stem cell growth factor-like mRNA. A ribozyme havingspecificity for a stem cell growth factor-like-encoding nucleic acid canbe designed based upon the nucleotide sequence of a stem cell growthfactor-like cDNA disclosed herein. For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a stem cell growth factor-like-encoding mRNA. See,e.g., U.S. Pat. No. 4,987,071 to Cech, et al. and U.S. Pat. No.5,116,742 to Cech, et al. Stem cell growth factor-like mRNA can also beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science261, 1411-1418.

Alternatively, stem cell growth factor-like gene expression can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the stem cell growth factor-like nucleic acid(e.g., the stem cell growth factor-like promoter and/or enhancers) toform triple helical structures that prevent transcription of the stemcell growth factor-like gene in target cells. See, e.g., Helene, 1991.Anticancer Drug Des. 6, 569-84; Helene, et al. 1992. Ann. N.Y. Acad.Sci. 660, 27-36; Maher, 1992. Bioassays 14, 807-15.

In various embodiments, the stem cell growth factor-like nucleic acidscan be modified at the base moiety, sugar moiety or phosphate backboneto improve, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids. See, e.g.,Hyrup, et al., 1996. Bioorg Med Chem 4, 5-23. As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics (e.g.,DNA mimics) in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup, et al.,1996. supra; Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93,14670-14675.

PNAs of stem cell growth factor-like can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,e.g., inducing transcription or translation arrest or inhibitingreplication. PNAs of stem cell growth factor-like can also be used, forexample, in the analysis of single base pair mutations in a gene (e.g.,PNA directed PCR clamping; as artificial restriction enzymes when usedin combination with other enzymes, e.g., S1 nucleases (see, Hyrup, etal., 1996. supra); or as probes or primers for DNA sequence andhybridization (see, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al.,1996. supra).

In another embodiment, PNAs of stem cell growth factor-like can bemodified, e.g., to enhance their stability or cellular uptake, byattaching lipophilic or other helper groups to PNA, by the formation ofPNA-DNA chimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. For example, PNA-DNA chimeras of stem cellgrowth factor-like can be generated that may combine the advantageousproperties of PNA and DNA. Such chimeras allow DNA recognition enzymes(e.g., RNase H and DNA polymerases) to interact with the DNA portionwhile the PNA portion would provide high binding affinity andspecificity. PNA-DNA chimeras can be linked using linkers of appropriatelengths selected in terms of base stacking, number of bonds between thenucleobases, and orientation (see, Hyrup, et al., 1996. supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup, etal., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24, 3357-3363.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA. See,e.g. Mag, et al., 1989. Nucl Acid Res 17, 5973-5988. PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment. See, e.g., Finn, et al., 1996.supra. Alternatively, chimeric molecules can be synthesized with a 5′DNA segment and a 3′ PNA segment. See, e.g., Petersen, et al., 1975.Bioorg. Med. Chem. Lett. 5, 1119-11124.

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci. U.S.A. 86,6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84, 648-652;PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g.,PCT Publication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krol,et al., 1988. BioTechniques 6, 958-976) or intercalating agents (see,e.g., Zon, 1988. Pharm. Res. 5, 539-549). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,a hybridization triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, and the like.

5.4 Hosts

The present invention further provides host cells genetically engineeredto contain the polynucleotides of the invention. For example, such hostcells may contain nucleic acids of the invention introduced into thehost cell using known transformation, transfection or infection methods.The present invention still further provides host cells geneticallyengineered to express the polynucleotides of the invention, wherein suchpolynucleotides are in operative association with a regulatory sequenceheterologous to the host cell which drives expression of thepolynucleotides in the cell.

Knowledge of stem cell growth factor-like DNA sequences allows formodification of cells to permit, or increase, expression of stem cellgrowth factor-like polypeptide. Cells can be modified (e.g., byhomologous recombination) to provide increased stem cell growthfactor-like polypeptide expression by replacing, in whole or in part,the naturally occurring stem cell growth factor-like promoter with allor part of a heterologous promoter so that the cells stem cell growthfactor-like polypeptide is expressed at higher levels. The heterologouspromoter is inserted in such a manner that it is operatively linked tostem cell growth factor-like encoding sequences. See, for example, PCTInternational Publication No. WO94/12650, PCT International PublicationNo. WO92/20808, and PCT International Publication No. WO91/09955. It isalso contemplated that, in addition to heterologous promoter DNA,amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CADgene which encodes carbamyl phosphate synthase, aspartatetranscarbamylase, and dihydroorotase) and/or intron DNA may be insertedalong with the heterologous promoter DNA. If linked to the stem cellgrowth factor-like coding sequence, amplification of the marker DNA bystandard selection methods results in co-amplification of the stem cellgrowth factor-like coding sequences in the cells.

The host cell can be a higher eukaryotic host cell, such as a mammaliancell, a lower eukaryotic host cell, such as a yeast cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the recombinant construct into the host cell can be effected bycalcium phosphate transfection, DEAE, dextran mediated transfection, orelectroporation (Davis, L. et al., Basic Methods in Molecular Biology(1986)). The host cells containing one of polynucleotides of theinvention, can be used in conventional manners to produce the geneproduct encoded by the isolated fragment (in the case of an ORF) or canbe used to produce a heterologous protein under the control of the EMF.

Any host/vector system can be used to express one or more of the ORFs ofthe present invention. These include, but are not limited to, eukaryotichosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9 cells, as wellas prokaryotic host such as E. coli and B. subtilis. The most preferredcells are those which do not normally express the particular polypeptideor protein or which expresses the polypeptide or protein at low naturallevel. Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell23:175 (1981), and other cell lines capable of expressing a compatiblevector, for example, the C127, 3T3, CHO, HeLa and BHK cell tines.Mammalian expression vectors will comprise an origin of replication, asuitable promoter, and also any necessary ribosome binding sites,polyadenylation site, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Recombinantpolypeptides and proteins produced in bacterial culture are usuallyisolated by initial extraction from cell pellets, followed by one ormore salting-out, aqueous ion exchange or size exclusion chromatographysteps. Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for final purification steps.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, humanepidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, othertransformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomzyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itmay be necessary to modify the protein produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

In another embodiment of the present invention, cells and tissues may beengineered to express an endogenous gene comprising the polynucleotidesof the invention under the control of inducible regulatory elements, inwhich case the regulatory sequences of the endogenous gene may bereplaced by homologous recombination. As described herein, genetargeting can be used to replace a gene's existing regulatory regionwith a regulatory sequence isolated from a different gene or a novelregulatory sequence synthesized by genetic engineering methods. Suchregulatory sequences may be comprised of promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, and regulatory protein binding sitesor combinations of said sequences. Alternatively, sequences which affectthe structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting, includingpolyadenylation signals, mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the host cell genome. Theidentification of the targeting event may also be facilitated by the useof one or more marker genes exhibiting the property of negativeselection, such that the negatively selectable marker is linked to theexogenous DNA, but configured such that the negatively selectable markerflanks the targeting sequence, and such that a correct homologousrecombination event with sequences in the host cell genome does notresult in the stable integration of the negatively selectable marker.Markers useful for this purpose include the Herpes Simplex Virusthymidine kinase (TK) gene or the bacterial xanthine-guaninephosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used inaccordance with this aspect of the invention are more particularlydescribed in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461to Sherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

5.5 Polypeptides of the Invention

The isolated polypeptides of the invention include, but are not limitedto, a polypeptide comprising: the amino acid sequence set forth as SEQID NO: 10, 13-24, 32 or 34 or an amino acid sequence encoded by any oneof the nucleotide sequences SEQ ID NO: 1-9, 11, 12, 31 or 33 or thecorresponding full length or mature protein. Polypeptides of theinvention also include polypeptides preferably with biological orimmunological activity that are encoded by: (a) a polynucleotide havingany one of the nucleotide sequences set forth in the SEQ ID NO: 1-9, 11,12, 31 or 33 or (b) polynucleotides encoding the amino acid sequence setforth as SEQ ID NO: 10, 13-24, 32 or 34 or (c) polynucleotides thathybridize to the complement of the polynucleotides of either (a) or (b)under stringent hybridization conditions. The invention also providesbiologically active or immunologically active variants of any of thepolypeptide amino acid sequences set forth as SEQ ID NO: 10, 13-24, 32or 32 or the corresponding full length or mature protein; and“substantial equivalents” thereof (e.g., with at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%,typically at least about 95%, 96%, 97%, more typically at least about98%, or most typically at least about 99% amino acid identity) thatretain biological activity. Polypeptides encoded by allelic variants mayhave a similar, increased, or decreased activity compared topolypeptides comprising SEQ ID NO: 10, 13-24, 32 or 34.

Protein compositions of the present invention may further comprise anacceptable carrier, such as a hydrophilic, e.g., pharmaceuticallyacceptable, carrier.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of host cells of the invention in asuitable culture medium, and purifying the protein from the cells or theculture in which the cells are grown. For example, the methods of theinvention include a process for producing a polypeptide in which a hostcell containing a suitable expression vector that includes apolynucleotide of the invention is cultured under conditions that allowexpression of the encoded polypeptide. The polypeptide can be recoveredfrom the culture, conveniently from the culture medium, or from a lysateprepared from the host cells and further purified. Preferred embodimentsinclude those in which the protein produced by such process is a fulllength or mature form of the protein.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” is intended nucleotide fragments which differ froma nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins. Avariety of methodologies known in the art can be utilized to obtain anyone of the isolated polypeptides or proteins of the present invention.At the simplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. This technique isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. In an alternative method, thepolypeptide or protein is purified from bacterial cells which naturallyproduce the polypeptide or protein. One skilled in the art can readilyfollow known methods for isolating polypeptides and proteins in order toobtain one of the isolated polypeptides or proteins of the presentinvention. These include, but are not limited to, immunochromatography,HPLC, size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel et al., CurrentProtocols in Molecular Biology. Polypeptide fragments that retainbiological/immunological activity include fragments encoding greaterthan about 100 amino acids, or greater than about 200 amino acids, andfragments that encode specific protein domains.

The polypeptides and proteins of the present invention can alternativelybe purified from cells which have been altered to express the desiredpolypeptide or protein. As used herein, a cell is said to be altered toexpress a desired polypeptide or protein when the cell, through geneticmanipulation, is made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.

The protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the protein.

The protein may also be produced by known conventional chemicalsynthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. Thus, they may be employedas biologically active or immunological substitutes for natural,purified proteins in screening of therapeutic compounds and inimmunological processes for the development of antibodies.

The proteins provided herein also include proteins characterized byamino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications in the peptide or DNA sequences can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein. Regions of the protein thatare important for the protein function can be determined by variousmethods known in the art including the alanine-scanning method whichinvolved systematic substitution of single or strings of amino acidswith alanine, followed by testing the resulting alanine-containingvariant for biological activity. This type of analysis determines theimportance of the substituted amino acid(s) in biological activity.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and areuseful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are encompassed by the present invention.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), incorporated herein byreference. As used herein, an insect cell capable of expressing apolynucleotide of the present invention is “transformed.”

The protein of the invention may be prepared by culturing transformedhost cells under culture conditions suitable to express the recombinantprotein. The resulting expressed protein may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the protein may also include anaffinity column containing agents which will bind to the protein; one ormore column steps over such affinity resins as concanavalin A-agarose,heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography.

Alternatively, the protein of the invention may also be expressed in aform which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a Histag. Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The proteincan also be tagged with an epitope and subsequently purified by using aspecific antibody directed to such epitope. One such epitope (“FLAG®”)is commercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an “isolatedprotein.”

The polypeptides of the invention include analogs (variants). Analogsembrace fragments, as well as antagonists which comprise one or moreamino acids deleted, inserted, or substituted. Analogs of the inventionalso embrace fusions of the polypeptide of the invention ormodifications of the polypeptide of the invention or analog is fused toanother moiety or moieties, e.g., targeting moiety, imaging moiety oranother therapeutic agent. Such analogs may exhibit improved propertiessuch as activity and/or stability. Examples of moieties which may befused to polypeptides of the invention or analogs thereof include, forexample, targeting moieties which provide for the delivery ofpolypeptide to desired cell types. Other moieties which may be fused tothe polypeptides of the invention include therapeutic agents which areused for treatment of disorders described herein.

5.5.1 Determining Polypeptide and Polynucleotide Identity and Similarity

Preferred identity and/or similarity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in computer programs including, but are notlimited to, the GCG program package, including GAP (Devereux, J., etal., Nucleic Acids Research 12(1): 387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA(Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST(Altschul S. F. et al., Nucleic Acids Res. vol. 25, pp. 3389-3402,herein incorporated by reference), the eMatrix software (Wu et al., J.Comp. Biol., vol. 6, pp. 219-235 (1999), herein incorporated byreference), eMotif software (Nevill-Manning et al, ISMB-97, vol 4, pp.202-209, herein incorporated by reference), the GeneAtlas software(Molecular Simulations Inc. (MSI), San Diego, Calif.) (Sanchez and Sali(1998) Proc. Natl. Acad. Sci., 95, 13597-13602; Kitson D H et al, (2000)“Remote homology detection using structural modeling—an evaluation”Submitted; Fischer and Eisenberg (1996) Protein Sci. 5, 947-955), NeuralNetwork SignalP V1.1 program (from Center for Biological SequenceAnalysis, The Technical University of Denmark) and the Kyte-Doolittlehydrophobocity prediction algorithm (J. Mol Biol, 157, pp. 105-31(1982), incorporated herein by reference). The BLAST programs arepublicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul, S., et al.NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol.215:403-410 (1990).

5.6 Chimeric and Fusion Proteins

The invention also provides stem cell growth factor-like chimeric orfusion proteins. As used herein, a stem cell growth factor-like“chimeric protein” or “fusion protein” comprises a stem cell growthfactor-like polypeptide operatively linked to either a different stemcell growth factor-like polypeptide or a non-stem cell growthfactor-like polypeptide. An “stem cell growth factor-like polypeptide”refers to a polypeptide having an amino acid sequence corresponding to astem cell growth factor-like protein, whereas a “non-stem cell growthfactor-like polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein that is not substantially homologousto the stem cell growth factor-like protein, e.g., a protein that isdifferent from the stem cell growth factor-like protein and that isderived from the same or a different organism. Within a stem cell growthfactor-like fusion protein the stem cell growth factor-like polypeptidecan correspond to all or a portion of a stem cell growth factor-likeprotein. In one embodiment, a stem cell growth factor-like fusionprotein comprises at least one biologically active portion of a stemcell growth factor-like protein. In another embodiment, a stem cellgrowth factor-like fusion protein comprises at least two biologicallyactive portions of a stem cell growth factor-like protein. In yetanother embodiment, a stem cell growth factor-like fusion proteincomprises at least three biologically active portions of a stem cellgrowth factor-like protein. Within the fusion protein, the term“operatively-linked” is intended to indicate that the stem cell growthfactor-like polypeptide(s) and/or the non-stem cell growth factor-likepolypeptide are fused in-frame with one another. The non-stem cellgrowth factor-like polypeptide can be fused to the N-terminus orC-terminus of the stem cell growth factor-like polypeptide.

In one embodiment, the fusion protein is a GST-stem cell growthfactor-like fusion protein in which the stem cell growth factor-likesequences are fused to the C-terminus of the GST (glutathioneS-transferase) sequences. Such fusion proteins can facilitate thepurification of recombinant stem cell growth factor-like polypeptides.

In another embodiment, the fusion protein is a stem cell growthfactor-like protein containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of stem cell growth factor-like can beincreased through use of a heterologous signal sequence.

In yet another embodiment, the fusion protein is a stem cell growthfactor-like-immunoglobulin fusion protein in which the stem cell growthfactor-like sequences are fused to sequences derived from a member ofthe immunoglobulin protein family. The stem cell growthfactor-like-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a stem cell growth factor-likeligand and a stem cell growth factor-like protein on the surface of acell, to thereby suppress stem cell growth factor-like-mediated signaltransduction in vivo. The stem cell growth factor-like-immunoglobulinfusion proteins can be used to affect the bioavailability of a stem cellgrowth factor-like cognate ligand. Inhibition of the stem cell growthfactor-like ligand/stem cell growth factor-like interaction can beuseful therapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g. promoting orinhibiting) cell survival. Moreover, the stem cell growthfactor-like-immunoglobulin fusion proteins of the invention can be usedas immunogens to produce anti-stem cell growth factor-like antibodies ina subject, to purify stem cell growth factor-like ligands, and inscreening assays to identify molecules that inhibit the interaction ofstem cell growth factor-like with a stem cell growth factor-like ligand.

Stem cell growth factor-like chimeric or fusion protein of the inventioncan be produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, e.g., byemploying blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers that give rise tocomplementary overhangs between two consecutive gene fragments that cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Ausubel, et al. (eds.) Current Protocols inMolecular Biology, John Wiley & Sons, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST polypeptide). Stem cell growth factor-like-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the stem cell growth factor-like protein.

5.7 Gene Therapy

Mutations in the polynucleotides of the invention gene may result inloss of normal function of the encoded protein. The invention thusprovides gene therapy to restore normal activity of the polypeptides ofthe invention; or to treat disease states involving polypeptides of theinvention. Delivery of a functional genes encoding polypeptides of theinvention to appropriate cells is effected ex vivo, in situ, or in vivoby use of vectors, and more particularly viral vectors (e.g.,adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by useof physical DNA transfer methods (e.g., liposomes or chemicaltreatments). See, for example, Anderson, Nature, supplement to vol. 392,no. 6679, pp.25-20 (1998). For additional reviews of gene therapytechnology see Friedmann, Science, 244: 1275-1281 (1989); Verma,Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460(1992). Introduction of any one of the nucleotides of the presentinvention or a gene encoding the polypeptides of the present inventioncan also be accomplished with extrachromosomal substrates (transientexpression) or artificial chromosomes (stable expression). Cells mayalso be cultured ex vivo in the presence of proteins of the presentinvention in order to proliferate or to produce a desired effect on oractivity in such cells. Treated cells can then be introduced in vivo fortherapeutic purposes. Alternatively, it is contemplated that in otherhuman disease states, preventing the expression of or inhibiting theactivity of polypeptides of the invention will be useful in treating thedisease states. It is contemplated that antisense therapy or genetherapy could be applied to negatively regulate the expression ofpolypeptides of the invention.

Other methods inhibiting expression of a protein include theintroduction of antisense molecules to the nucleic acids of the presentinvention, their complements, or their translated RNA sequences, bymethods known in the art, the removal of the nucleic acids of thepresent invention such as using targeted deletion methods, or theinsertion of a negative regulatory element such as a silencer, which istissue specific. Further, the polypeptides of the present invention canbe inhibited by the introduction of antisense molecules that hybridizeto nucleic acids that encode for the polypeptides of the presentinvention and by the removal of a gene that encode for the polypeptidesof the present invention.

The present invention still further provides cells geneticallyengineered in vivo to express the polynucleotides of the invention,wherein such polynucleotides are in operative association with aregulatory sequence heterologous to the host cell which drivesexpression of the polynucleotides in the cell. These methods can be usedto increase or decrease the expression of the polynucleotides of thepresent invention.

Knowledge of DNA sequences provided by the invention allows formodification of cells to permit, increase, or decrease, expression ofendogenous polypeptide. Cells can be modified (e.g., by homologousrecombination) to provide increased polypeptide expression by replacing,in whole or in part, the naturally occurring promoter with all or partof a heterologous promoter so that the cells express the protein athigher levels. The heterologous promoter is inserted in such a mannerthat it is operatively linked to the desired protein encoding sequences.See, for example, PCT International Publication No. WO 94/12650, PCTInternational Publication No. WO 92/20808, and PCT InternationalPublication No. WO 91/09955. It is also contemplated that, in additionto heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr,and the multifunctional CAD gene which encodes carbamyl phosphatesynthase, aspartate transcarbamylase, and dihydroorotase) and/or intronDNA may be inserted along with the heterologous promoter DNA. If linkedto the desired protein coding sequence, amplification of the marker DNAby standard selection methods results in co-amplification of the desiredprotein coding sequences in the cells.

In another embodiment of the present invention, cells and tissues may beengineered to express an endogenous gene comprising the polynucleotidesof the invention under the control of inducible regulatory elements, inwhich case the regulatory sequences of the endogenous gene may bereplaced by homologous recombination. As described herein, genetargeting can be used to replace a gene's existing regulatory regionwith a regulatory sequence isolated from a different gene or a novelregulatory sequence synthesized by genetic engineering methods. Suchregulatory sequences may be comprised of promoters, enhancers,scaffold-attachment regions, negative regulatory elements,transcriptional initiation sites, regulatory protein binding sites orcombinations of said sequences. Alternatively, sequences which affectthe structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting. Thesesequence include polyadenylation signals, mRNA stability elements,splice sites, leader sequences for enhancing or modifying transport orsecretion properties of the protein, or other sequences which alter orimprove the function or stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the cell genome. The identification ofthe targeting event may also be facilitated by the use of one or moremarker genes exhibiting the property of negative selection, such thatthe negatively selectable marker is linked to the exogenous DNA, butconfigured such that the negatively selectable marker flanks thetargeting sequence, and such that a correct homologous recombinationevent with sequences in the host cell genome does not result in thestable integration of the negatively selectable marker. Markers usefulfor this purpose include the Herpes Simplex Virus thymidine kinase (TK)gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)gene.

The gene targeting or gene activation techniques which can be used inaccordance with this aspect of the invention are more particularlydescribed in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461to Sherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

5.8 Transgenic Animals

In preferred methods to determine biological functions of thepolypeptides of the invention in vivo, one or more genes provided by theinvention are either over expressed or inactivated in the germ line ofanimals using homologous recombination [Capecchi, Science 244:1288-1292(1989)]. Animals in which the gene is over expressed, under theregulatory control of exogenous or endogenous promoter elements, areknown as transgenic animals. Animals in which an endogenous gene hasbeen inactivated by homologous recombination are referred to as“knockout” animals. Knockout animals, preferably non-human mammals, canbe prepared as described in U.S. Pat. No. 5,557,032, incorporated hereinby reference. Transgenic animals are useful to determine the rolespolypeptides of the invention play in biological processes, andpreferably in disease states. Transgenic animals are useful as modelsystems to identify compounds that modulate lipid metabolism. Transgenicanimals, preferably non-human mammals, are produced using methods asdescribed in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122,incorporated herein by reference.

Transgenic animals can be prepared wherein all or part of apolynucleotides of the invention promoter is either activated orinactivated to alter the level of expression of the polypeptides of theinvention. Inactivation can be carried out using homologousrecombination methods described above. Activation can be achieved bysupplementing or even replacing the homologous promoter to provide forincreased protein expression. The homologous promoter can besupplemented by insertion of one or more heterologous enhancer elementsknown to confer promoter activation in a particular tissue.

5.9 Uses and Biological Activity of Stem Cell Growth Factor-LikePolypeptide

Stem cell growth factor-like polypeptide is based on polynucleotidesisolated from cDNA libraries prepared from human testis cells (Hyseqclone identification numbers 2880984 and 2881695), from human fetal skin(Hyseq clone identification number 15375176), adult spleen (Hyseq cloneidentification number 14856094), and human endothelial cells (Hyseqclone identification numbers 13804756, 13687487, 13804756).

FIG. 1 shows the alignment of polynucleotide SEQ ID NO: 9 and ESTsequences SEQ ID NO: 1-7. The nucleic acid sequences of the presentinvention (SEQ ID NO: 1-9) are expected encode polypeptides having stemcell growth factor activity, including hematopoietic stem cell growthfactor activity, as described herein. The polypeptide of SEQ ID NO: 10,fragments thereof, sequences having at least 90% homology, are alsoexpected to have stem cell growth factor activity, includinghematopoietic stem cell growth factor activity, as described herein.

The stem cell growth factor-like polypeptide of SEQ ID NO: 10 is anapproximately 272-amino acid protein with a predicted molecular mass ofapproximately 30 kDa unglycosylated. Protein database searches with theBLASTX algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300 (1993)and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 10 is homologous tothrombospondin type I domain and a human secreted protein clone da228_(—)6. Protein database search with eMATRIX software (StanfordUniversity, Stanford Calif.) further show that a portion of SEQ ID NO:10 has a laminin-type EGF-like (LE) domain, a vertebratemetallothioneins domain, an endogenous opioids neuropeptides precursorsproteins domain, a membrane attack complex components/perforin proteinsdomain, an HMG-I and HMG-Y DNA-binding domain proteins (Ahook), anHMG1/2 protein domain, a vertebrate metallothionein signature domain,and a neurohypophysial hormone signature domain.

A predicted approximately twenty-one residue signal peptide is encodedfrom approximately residue 1 to residue 21 of SEQ ID NO: 10 (SEQ ID NO:15). The extracellular portion is useful on its own. This can beconfirmed by expression in mammalian cells and sequencing of the cleavedproduct. The signal peptide region was predicted using Neural NetworkSignalP V1.1 program (Nielsen et al, (1997) Int. J. Neural Syst. 8,581-599). One of skill in the art will recognize that the actualcleavage site may be different than that predicted by the computerprogram. SEQ ID NO: 16 is the peptide resulting when the predictedsignal peptide is removed from SEQ ID NO: 10.

Using eMATRIX software package (Stanford University, Stanford, Calif.)(Wu et al., J. Comp. Biol., vol. 6, pp. 219-235 (1999), hereinincorporated by reference), Siglec-like polypeptide of SEQ ID NO: 10 isexpected to have following domains, wherein A=Alanine, C=Cysteine,D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine,H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine: Laminin-type EGF-like (LE) domainproteins at 100 ADCDTCFNKNFCTKCKSGFYLHL 122 (SEQ ID NO: 17)

Vertebrate metallothioneins proteins at (SEQ ID NO: 18) 92INKCTKCKADCDTCFNKNFCTKCKSGFYLHLGKCLDNCPEGLEANN 137

Endogenous opioids neuropeptides precursors proteins at 33MHPNVSQGCQGGCATCSDYN 52 (SEQ ID NO: 19)

Membrane attack complex components/perforin proteins at 145IVHCEVSEWNPWSPCTKKGKTCGFKRGTET (SEQ ID NO: 20) RVREIIQ 181

HMG-I and HMG-Y DNA-binding domain proteins (Ahook) at 213 KKGRERKRKK222 (SEQ ID NO: 21)

HMG1/2 proteins at 198 KCTVQRKKCQKGERGKKGRERKRKKPNKGE (SEQ ID NO: 22)SKEAIPDSKSLE 239

VERTEBRATE METALLOTHIONEIN SIGNATURE at 104 TCFNKNFCTKCKSG 117 (SEQ IDNO: 23)

NEUROHYPOPHYSIAL HORMONE SIGNATURE at 148 CEVSEWNPWSPCTKKGKTCG 167 (SEQID NO: 24)

Motif 100-122, a laminin-type EGF-like domain, is a component ofextracellular matrix which promotes cell growth. The membrane attackcomplex component/perforin domain (145-185) is postulated to mediatecell-cell interaction and thus cell growth and differentiation.Neurohypophysial hormone is itself regulated by many other factorsincluding Interleukin-1 beta and Interleukin-6. The presence of thesemotifs are expected in stem cell growth factor activity.

Stem cell growth factor-like protein and/or fragments or derivativeswould have similar activity to stem cell growth factors and anabolicgrowth factors and receptors.

Polypeptides of the invention having stem cell growth factor-likeactivity are useful for but not limited to cell growth andmorphogenesis, including hematopoietic stem cell growth and/or growth ofa particular hematopoietic cell type (such as B or T cells), tissuespecific stem cell growth, epithelial cell growth and regulation,ovarian follicle development, promoting nerve cell growth, sustainingneuronal populations, cartilage remodeling, wound repair, bone growth,immunosuppression, immune response modulation, modulating antibody andcell mediated immunity and vascular remodeling. The polypeptides of theinvention can therefore be employed in but not limited to theprophylaxis or treatment of disorders and diseases caused by orinvolving wound healing, growth and development, regulation of cartilagegrowth and development, vascular remodeling (angiogenesis),immunosuppression, follicle growth and development and neurite growthand development. Polypeptides of the invention can also be used in theproduction of and maintenance of transplants or epidermal grafts.

The polynucleotides and proteins of the present invention are expectedto exhibit one or more of the uses or biological activities (includingthose associated with assays cited herein) identified herein. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins or of polynucleotidesencoding such proteins (such as, for example, in gene therapies orvectors suitable for introduction of DNA). The mechanism underlying theparticular condition or pathology will dictate whether the polypeptidesof the invention, the polynucleotides of the invention or modulators(activators or inhibitors) thereof would be beneficial to the subject inneed of treatment. Thus, “therapeutic compositions of the invention”include compositions comprising isolated polynucleotides (includingrecombinant DNA molecules, cloned genes and degenerate variants thereof)or polypeptides of the invention (including full length protein, matureprotein and truncations or domains thereof), or compounds and othersubstances that modulate the overall activity of the target geneproducts, either at the level of target gene/protein expression ortarget protein activity. Such modulators include polypeptides, analogs,(variants), including fragments and fusion proteins, antibodies andother binding proteins; chemical compounds that directly or indirectlyactivate or inhibit the polypeptides of the invention (identified, e.g.,via drug screening assays as described herein); antisensepolynucleotides and polynucleotides suitable for triple helix formation;and in particular antibodies or other binding partners that specificallyrecognize one or more epitopes of the polypeptides of the invention.

The protein of the present invention may likewise be involved incellular activation or in one of the other physiological pathwaysdescribed herein.

5.9.1 Research Uses and Utilities

The polynucleotides provided by the present invention can be used by theresearch community for various purposes. The polynucleotides can be usedto express recombinant protein for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingprotein is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates); as molecular weight markers on gels; as chromosome markers ortags (when labeled) to identify chromosomes or to map related genepositions; to compare with endogenous DNA sequences in patients toidentify potential genetic disorders; as probes to hybridize and thusdiscover novel, related DNA sequences; as a source of information toderive PCR primers for genetic fingerprinting; as a probe to“subtract-out” known sequences in the process of discovering other novelpolynucleotides; for selecting and making oligomers for attachment to a“gene chip” or other support, including for examination of expressionpatterns; to raise anti-protein antibodies using DNA immunizationtechniques; and as an antigen to raise anti-DNA antibodies or elicitanother immune response. Where the polynucleotide encodes a proteinwhich binds or potentially binds to another protein (such as, forexample, in a receptor-ligand interaction), the polynucleotide can alsobe used in interaction trap assays (such as, for example, that describedin Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotidesencoding the other protein with which binding occurs or to identifyinhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used inassays to determine biological activity, including in a panel ofmultiple proteins for high-throughput screening; to raise antibodies orto elicit another immune response; as a reagent (including the labeledreagent) in assays designed to quantitatively determine levels of theprotein (or its receptor) in biological fluids; as markers for tissuesin which the corresponding protein is preferentially expressed (eitherconstitutively or at a particular stage of tissue differentiation ordevelopment or in a disease state); and, of course, to isolatecorrelative receptors or ligands. Where the protein binds or potentiallybinds to another protein (such as, for example, in a receptor-ligandinteraction), the protein can be used to identify the other protein withwhich binding occurs or to identify inhibitors of the bindinginteraction. Proteins involved in these binding interactions can also beused to screen for peptide or small molecule inhibitors or agonists ofthe binding interaction.

The polypeptides of the invention are also useful for making antibodysubstances that are specifically immunoreactive with stem cell growthfactor-like proteins. Antibodies and portions thereof (e.g., Fabfragments) which bind to the polypeptides of the invention can be usedto identify the presence of such polypeptides in a sample. For example,the level of the native protein corresponding to SEQ ID NO: 10 in atissue sample can be determined as an indication of chrondrocytedifferentiation or embryonic status. Such determinations are carried outusing any suitable immunoassay format, and any polypeptide of theinvention that is specifically bound by the antibody can be employed asa positive control.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold SpringHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

5.9.2 Nutritional Uses

Polynucleotides and proteins of the present invention can also be usedas nutritional sources or supplements. Such uses include withoutlimitation use as a protein or amino acid supplement, use as a carbonsource, use as a nitrogen source and use as a source of carbohydrate. Insuch cases the protein or polynucleotide of the invention can be addedto the feed of a particular organism or can be administered as aseparate solid or liquid preparation, such as in the form of powder,pills, solutions, suspensions or capsules. In the case ofmicroorganisms, the protein or polynucleotide of the invention can beadded to the medium in or on which the microorganism is cultured.

Additionally, the polypeptides of the invention can be used as molecularweight markers, and as a food supplement. A polypeptide consisting ofSEQ ID NO: 10, for example, has a molecular mass of approximately 30 kDain its unprocessed and unglycosylated state. Protein food supplementsare well known and the formulation of suitable food supplementsincluding polypeptides of the invention is within the level of skill inthe food preparation art.

5.9.3 Cytokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit activity relating tocytokine, cell proliferation (either inducing or inhibiting) or celldifferentiation (either inducing or inhibiting) activity or may induceproduction of other cytokines in certain cell populations. Apolynucleotide of the invention can encode a polypeptide exhibiting suchattributes. Many protein factors discovered to date, including all knowncytokines, have exhibited activity in one or more factor-dependent cellproliferation assays, and hence the assays serve as a convenientconfirmation of cytokine activity. The activity of therapeuticcompositions of the present invention is evidenced by any one of anumber of routine factor dependent cell proliferation assays for celllines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1,Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the inventioncan be used in the following:

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I.Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human interleukin-, Schreiber, R. D. In CurrentProtocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8,John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E.Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991;Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai etal., J. Immunol. 140:508-512, 1988.

5.9.4 Stem Cell Growth Factor Activity

Polypeptides of the present invention have been shown to exhibit stemcell growth factor activity and to be involved in the proliferation,differentiation and survival of pluripotent and totipotent stem cellsincluding primordial germ cells, embryonic stem cells, neural stemcells, skeletal muscle stem cells, mesencymal stem cells, hematopoieticstem cells and/or germ line stem cells. Administration of thepolypeptide of the invention to stem cells in vivo or ex vivo maintainsand expands cell populations in a totipotential or pluripotential statewhich would be useful for re-engineering damaged or diseased tissues,transplantation including solid organs and bone marrow transplants,manufacture of bio-pharmaceuticals and the development of bio-sensors.The ability to produce large quantities of human cells has importantworking applications for the production of human proteins whichcurrently must be obtained from non-human sources or donors,implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such asbone marrow, skin, cartilage, tendons, bone, muscle (including cardiacmuscle), blood vessels, cornea, neural cells, gastrointestinal cells andothers; and organs for transplantation such as kidney, liver, pancreas(including islet cells), heart and lung.

It is contemplated that multiple different exogenous growth factorsand/or cytokines may be administered in combination with the polypeptideof the invention to achieve the desired effect, including any of thegrowth factors listed herein, other stem cell maintenance factors, andspecifically including stem cell factor (SCF), leukemia inhibitoryfactor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins,recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatoryprotein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO),platelet factor 4 (PF4), platelet-derived growth factor (PDGF), neuralgrowth factors and basic fibroblast growth factor (bFGF).

Since totipotent stem cells can give rise to virtually any mature celltype, expansion of these cells in culture will facilitate the productionof large quantities of mature cells. Techniques for culturing stem cellsare known in the art and administration of polypeptides of theinvention, optionally with other growth factors and/or cytokines, isexpected to enhance the survival and proliferation of the stem cellpopulations. This can be accomplished by direct administration of thepolypeptide of the invention to the culture medium. Alternatively,stroma cells transfected with a polynucleotide that encodes for thepolypeptide of the invention can be used as a feeder layer for the stemcell populations in culture or in vivo. Stromal support cells for feederlayers may include embryonic bone marrow fibroblasts, bone marrowstromal cells, fetal liver cells, or cultured embryonic fibroblasts (seeU.S. Pat. No. 5,690,926).

Stem cells themselves can be transfected with a polynucleotide of theinvention to induce autocrine expression of the polypeptide of theinvention. This will allow for generation of undifferentiatedtotipotential/pluripotential stem cell lines that are useful as is orthat can then be differentiated into the desired mature cell types.These stable cell lines can also serve as a source of undifferentiatedtotipotential/pluripotential mRNA to create cDNA libraries and templatesfor polymerase chain reaction experiments. These studies would allow forthe isolation and identification of differentially expressed genes instem cell populations that regulate stem cell proliferation and/ormaintenance.

Expansion and maintenance of totipotent stem cell populations is usefulin the treatment of many pathological conditions. For example,polypeptides of the present invention may be used to manipulate stemcells in culture to give rise to neuroepithelial cells that can be usedto augment or replace cells damaged by illness, autoimmune disease,accidental damage or genetic disorders, inflammatory disease,immunodeficiency, leukemia and neoplastic myeloid disorders. Thepolypeptide of the invention can be useful for inducing theproliferation of neural cells and for the regeneration of nerve andbrain tissue, i.e. for the treatment of central and peripheral nervoussystem diseases and neuropathies, as well as mechanical and traumaticdisorders which involve degeneration, death or trauma to neural cells ornerve tissue. In addition, the expanded stem cell populations can alsobe genetically altered for gene therapy purposes and to decrease hostrejection of replacement tissues after grafting or implantation. Thepolypeptide of the invention can also be useful for inducing theproliferation of cardiac stem cells and for regenerating functionalheart tissue following cardiac damage induced by cardiac disorders suchas myocardial infarctions and artery blockage. In addition, thepolypeptides of the invention may strengthen cardiac muscle cells andprevent and/or repair the heart tissue damage due to heart failure. SeeWeismann, Science, 287: 1442-1446, 2001; Vogel, Science, 290: 1672-1674,2000 Kajstura et al., Nature, 410: 701-705, 2001.

Expression of the polypeptide of the invention and its effect on stemcells can also be manipulated to achieve controlled differentiation ofthe stem cells into more differentiated cell types. A broadly applicablemethod of obtaining pure populations of a specific differentiated celltype from undifferentiated stem cell populations involves the use of acell-type specific promoter driving a selectable marker. The selectablemarker allows only cells of the desired type to survive. For example,stem cells can be induced to differentiate into cardiomyocytes (Wobus etal., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L.W. In: Principles of Tissue Engineering eds. Lanza et al., AcademicPress (1997)). Alternatively, directed differentiation of stem cells canbe accomplished by culturing the stem cells in the presence of adifferentiation factor such as retinoic acid and an antagonist of thepolypeptide of the invention which would inhibit the effects ofendogenous stem cell factor activity and allow differentiation toproceed.

In vitro cultures of stem cells can be used to determine if thepolypeptide of the invention exhibits stem cell growth factor activity.Stem cells are isolated from any one of various cell sources (includinghematopoietic stem cells and embryonic stem cells) and cultured on afeeder layer, as described by Thompson et al. Proc. Natl. Acad. Sci,U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of theinvention alone or in combination with other growth factors orcytokines. The ability of the polypeptide of the invention to inducestem cells proliferation is determined by colony formation on semi-solidsupport e.g. as described by Bernstein et al., Blood, 77: 2316-2321(1991).

5.9.5 Hematopoiesis Regulating Activity

A protein of the present invention may be involved in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell disorders. Even marginal biological activity in support of colonyforming cells or of factor-dependent cell lines indicates involvement inregulating hematopoiesis, e.g. in supporting the growth andproliferation of erythroid progenitor cells alone or in combination withother cytokines, thereby indicating utility, for example, in treatingvarious anemias or for use in conjunction with irradiation/chemotherapyto stimulate the production of erythroid precursors and/or erythroidcells; in supporting the growth and proliferation of myeloid cells suchas granulocytes and monocytes/macrophages (i.e., traditional circulatingsoluble factor activity) useful, for example, in conjunction withchemotherapy to prevent or treat consequent myelo-suppression; insupporting the growth and proliferation of megakaryocytes andconsequently of platelets thereby allowing prevention or treatment ofvarious platelet disorders such as thrombocytopenia, and generally foruse in place of or complimentary to platelet transfusions; and/or insupporting the growth and proliferation of hematopoietic stem cellswhich are capable of maturing to any and all of the above-mentionedhematopoietic cells and therefore find therapeutic utility in variousstem cell disorders (such as those usually treated with transplantation,including, without limitation, aplastic anemia and paroxysmal nocturnalhemoglobinuria), as well as in repopulating the stem cell compartmentpost irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., inconjunction with bone marrow transplantation or with peripheralprogenitor cell transplantation (homologous or heterologous)) as normalcells or genetically manipulated for gene therapy.

Therapeutic compositions of the invention can be used in the following:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al.,Molecular and Cellular Biology 13:473486, 1993; McClanahan et al., Blood81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

5.9.6 Tissue Growth Activity

A protein of the present invention also may be involved in bone,cartilage, tendon, ligament and/or nerve tissue growth or regeneration,as well as in wound healing and tissue repair and replacement, and inhealing of burns, incisions and ulcers.

For example, induction of cartilage and/or bone growth in circumstanceswhere bone is not normally formed, has application in the healing ofbone fractures and cartilage damage or defects in humans and otheranimals. Compositions of a protein, antibody, binding partner, or othermodulator of the invention may have prophylactic use in closed as wellas open fracture reduction and also in the improved fixation ofartificial joints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

A protein of this invention may also be involved in attractingbone-forming cells, stimulating growth of bone-forming cells, orinducing differentiation of progenitors of bone-forming cells. Treatmentof osteoporosis, osteoarthritis, bone degenerative disorders, orperiodontal disease, such as through stimulation of bone and/orcartilage repair or by blocking inflammation or processes of tissuedestruction (collagenase activity, osteoclast activity, etc.) mediatedby inflammatory processes may also be possible using the composition ofthe invention.

Another category of tissue regeneration activity that may involve theprotein of the present invention is tendon/ligament formation. Inductionof tendon/ligament-like tissue or other tissue formation incircumstances where such tissue is not normally formed, has applicationin the healing of tendon or ligament tears, deformities and other tendonor ligament defects in humans and other animals. Such a preparationemploying a tendon/ligament-like tissue inducing protein may haveprophylactic use in preventing damage to tendon or ligament tissue, aswell as use in the improved fixation of tendon or ligament to bone orother tissues, and in repairing defects to tendon or ligament tissue. Denovo tendon/ligament-like tissue formation induced by a composition ofthe present invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendinitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The compositions of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a composition may be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a composition of the invention.

Compositions of the invention may also be useful to promote better orfaster closure of non-healing wounds, including without limitationpressure ulcers, ulcers associated with vascular insufficiency, surgicaland traumatic wounds, and the like.

Compositions of the present invention may also be involved in thegeneration or regeneration of other tissues, such as organs (including,for example, pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac) and vascular (including vascularendothelium) tissue, or for promoting the growth of cells comprisingsuch tissues. Inhibition or modulation of fibrotic scarring may allownormal tissue to regenerate.

A composition of the present invention may also be useful for gutprotection or regeneration and treatment of lung or liver fibrosis,reperfusion injury in various tissues, and conditions resulting fromsystemic cytokine damage.

A composition of the present invention may also be useful for promotingor inhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove.

Therapeutic compositions of the invention can be used in the following:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H.I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago,as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84(1978).

5.9.7 Immune Stimulating or Suppressing Activity

Compositions of the present invention may also exhibit immunestimulating or immune suppressing activity, including without limitationthe activities for which assays are described herein. A polynucleotideof the invention can encode a polypeptide involved in such activities. Aprotein or antibody, other binding partner, or other modulator of theinvention may be useful in the treatment of various immune deficienciesand disorders (including severe combined immunodeficiency (SCID)), e.g.,in regulating (up or down) growth and proliferation of T and/or Blymphocytes, as well as effecting the cytolytic activity of NK cells andother cell populations. These immune deficiencies may be genetic or becaused by viral (e.g., HIV) as well as bacterial or fungal infections,or may result from autoimmune disorders. More specifically, infectiousdiseases caused by viral, bacterial, fungal or other infection may betreatable using a protein, antibody, binding partner, or other modulatorof the invention, including infections by HIV, hepatitis viruses,herpesviruses, mycobacteria, Leishmania spp., malaria spp. and variousfungal infections such as candidiasis, as well as other conditions wherea boost to the immune system generally may be desirable, e.g., in thetreatment of cancer.

Autoimmune disorders which may involve a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein of the present invention may also to be involved in allergicreactions and conditions, such as asthma (particularly allergic asthma)or other respiratory problems.

Using the proteins, antibody, binding partners, or other modulators ofthe invention it may also be possible to modulate immune responses, in anumber of ways. The immune response may be enhanced or suppressed. Downregulation may be in the form of inhibiting or blocking an immuneresponse already in progress or may involve preventing the induction ofan immune response. The functions of activated T cells may be inhibitedby suppressing T cell responses or by inducing specific tolerance in Tcells, or both. Immunosuppression of T cell responses is generally anactive, non-antigen-specific, process which requires continuous exposureof the T cells to the suppressive agent. Tolerance, which involvesinducing non-responsiveness or anergy in T cells, is distinguishablefrom immunosuppression in that it is generally antigen-specific andpersists after exposure to the tolerizing agent has ceased.Operationally, tolerance can be demonstrated by the lack of a T cellresponse upon reexposure to specific antigen in the absence of thetolerizing agent.

Down regulating or preventing the immune response, e.g., preventing highlevel lymphokine synthesis by activated T cells, will be useful insituations of tissue, skin and organ transplantation and ingraft-versus-host disease (GVHD). For example, blockage of T cellfunction should result in reduced tissue destruction in tissuetransplantation. Typically, in tissue transplants, rejection of thetransplant is initiated through its recognition as foreign by T cells,followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks the immuneresponse (e.g. a receptor fragment, binding partner, or other modulatorsuch as antisense polynucleotides) may act as an immunosuppressant.

The efficacy of particular immune response modulators in preventingorgan transplant rejection or GVHD can be assessed using animal modelsthat are predictive of efficacy in humans. Examples of appropriatesystems which can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of blocking B lymphocyte antigen function in vivo on thedevelopment of that disease.

Blocking the inflammatory response may also be therapeutically usefulfor treating autoimmune diseases. Many autoimmune disorders are theresult of inappropriate activation of T cells that are reactive againstself tissue and which promote the production of cytokines andautoantibodies involved in the pathology of the diseases. Preventing theactivation of autoreactive T cells may reduce or eliminate diseasesymptoms. Administration of reagents which block costimulation of Tcells can be used to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of immune responses, may also be useful in therapy.Upregulation of immune responses may be in the form of enhancing anexisting immune response or eliciting an initial immune response. Forexample, enhancing an immune response may be useful in cases of viralinfection such as influenza, the common cold, and encephalitis.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro and reintroducing the in vitro activated T cells intothe patient.

The activity of therapeutic compositions of the invention may, amongother means, be measured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol.137:3494-3500, 1986; Bowman et al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W. Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:40624069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad. Sci. USA 88:7548-7551, 1991.

5.9.8 Activin/Inhibin Activity

A protein of the present invention may also exhibit activin- orinhibin-related activities. A polynucleotide of the invention may encodea polypeptide exhibiting such characteristics. Inhibins arecharacterized by their ability to inhibit the release of folliclestimulating hormone (FSH), while activins and are characterized by theirability to stimulate the release of follicle stimulating hormone (FSH).Thus, a protein of the present invention, alone or in heterodimers witha member of the inhibin family, may be useful as a contraceptive basedon the ability of inhibins to decrease fertility in female mammals anddecrease spermatogenesis in male mammals. Administration of sufficientamounts of other inhibins can induce infertility in these mammals.Alternatively, the protein of the invention, as a homodimer or as aheterodimer with other protein subunits of the inhibin group, may beuseful as a fertility inducing therapeutic, based upon the ability ofactivin molecules in stimulating FSH release from cells of the anteriorpituitary. See, for example, U.S. Pat. No. 4,798,885. A protein of theinvention may also be useful for advancement of the onset of fertilityin sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as, but not limitedto, cows, sheep and pigs.

The activity of a protein of the invention may, among other means, bemeasured by the following methods.

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al.,Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Masonet al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci.USA 83:3091-3095, 1986.

5.9.9 Chemotactic/Chemokinetic Activity

A protein of the present invention may be involved in chemotactic orchemokinetic activity for mammalian cells, including, for example,monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,epithelial and/or endothelial cells. A polynucleotide of the inventioncan encode a polypeptide exhibiting such attributes. Chemotactic andchemokinetic receptor activation can be used to mobilize or attract adesired cell population to a desired site of action. Chemotactic orchemokinetic compositions (e.g. proteins, antibodies, binding partners,or modulators of the invention) provide particular advantages intreatment of wounds and other trauma to tissues, as well as in treatmentof localized infections. For example, attraction of lymphocytes,monocytes or neutrophils to tumors or sites of infection may result inimproved immune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cellpopulation if it can stimulate, directly or indirectly, the directedorientation or movement of such cell population. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells. Whether a particular protein has chemotactic activity for apopulation of cells can be readily determined by employing such proteinor peptide in any known assay for cell chemotaxis.

Therapeutic compositions of the invention can be used in the following:

Assays for chemotactic activity (which will identify proteins thatinduce or prevent chemotaxis) consist of assays that measure the abilityof a protein to induce the migration of cells across a membrane as wellas the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol.25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnstonet al. J. of Immunol. 153:1762-1768, 1994.

5.9.10 Hemostatic and Thrombolytic Activity

A protein of the invention may also be involved in hemostatis orthrombolysis or thrombosis. A polynucleotide of the invention can encodea polypeptide exhibiting such attributes. Compositions may be useful intreatment of various coagulation disorders (including hereditarydisorders, such as hemophilias) or to enhance coagulation and otherhemostatic events in treating wounds resulting from trauma, surgery orother causes. A composition of the invention may also be useful fordissolving or inhibiting formation of thromboses and for treatment andprevention of conditions resulting therefrom (such as, for example,infarction of cardiac and central nervous system vessels (e.g., stroke).

Therapeutic compositions of the invention can be used in the following:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467474, 1988.

5.9.11 Cancer Diagnosis and Therapy

Polypeptides of the invention may be involved in cancer cell generation,proliferation or metastasis. Detection of the presence or amount ofpolynucleotides or polypeptides of the invention may be useful for thediagnosis and/or prognosis of one or more types of cancer. For example,the presence or increased expression of a polynucleotide/polypeptide ofthe invention may indicate a hereditary risk of cancer, a precancerouscondition, or an ongoing malignancy. Conversely, a defect in the gene orabsence of the polypeptide may be associated with a cancer condition.Identification of single nucleotide polymorphisms associated with canceror a predisposition to cancer may also be useful for diagnosis orprognosis.

Cancer treatments promote tumor regression by inhibiting tumor cellproliferation, inhibiting angiogenesis (growth of new blood vessels thatis necessary to support tumor growth) and/or prohibiting metastasis byreducing tumor cell motility or invasiveness. Therapeutic compositionsof the invention may be effective in adult and pediatric oncologyincluding in solid phase tumors/malignancies, locally advanced tumors,human soft tissue sarcomas, metastatic cancer, including lymphaticmetastases, blood cell malignancies including multiple myeloma, acuteand chronic leukemias, and lymphomas, head and neck cancers includingmouth cancer, larynx cancer and thyroid cancer, lung cancers includingsmall cell carcinoma and non-small cell cancers, breast cancersincluding small cell carcinoma and ductal carcinoma, gastrointestinalcancers including esophageal cancer, stomach cancer, colon cancer,colorectal cancer and polyps associated with colorectal neoplasia,pancreatic cancers, liver cancer, urologic cancers including bladdercancer and prostate cancer, malignancies of the female genital tractincluding ovarian carcinoma, uterine (including endometrial) cancers,and solid tumor in the ovarian follicle, kidney cancers including renalcell carcinoma, brain cancers including intrinsic brain tumors,neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cellinvasion in the central nervous system, bone cancers including osteomas,skin cancers including malignant melanoma, tumor progression of humanskin keratinocytes, squamous cell carcinoma, basal cell carcinoma,hemangiopericytoma and Karposi's sarcoma.

Polypeptides, polynucleotides, or modulators of polypeptides of theinvention (including inhibitors and stimulators of the biologicalactivity of the polypeptide of the invention) may be administered totreat cancer. Therapeutic compositions can be administered intherapeutically effective dosages alone or in combination with adjuvantcancer therapy such as surgery, chemotherapy, radiotherapy,thermotherapy, and laser therapy, and may provide a beneficial effect,e.g. reducing tumor size, slowing rate of tumor growth, inhibitingmetastasis, or otherwise improving overall clinical condition, withoutnecessarily eradicating the cancer.

The composition can also be administered in therapeutically effectiveamounts as a portion of an anti-cancer cocktail. An anti-cancer cocktailis a mixture of the polypeptide or modulator of the invention with oneor more anti-cancer drugs in addition to a pharmaceutically acceptablecarrier for delivery. The use of anti-cancer cocktails as a cancertreatment is routine. Anti-cancer drugs that are well known in the artand can be used as a treatment in combination with the polypeptide ormodulator of the invention include: Actinomycin D, Aminoglutethimide,Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl(Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl,Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213),Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, InterferonAlpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine,Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine,Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide,Plicamycin, Procarbazine HCl, Streptdzocin, Tamoxifen citrate,Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate,Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone,Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

In addition, therapeutic compositions of the invention may be used forprophylactic treatment of cancer. There are hereditary conditions and/orenvironmental situations (e.g. exposure to carcinogens) known in the artthat predispose an individual to developing cancers. Under thesecircumstances, it may be beneficial to treat these individuals withtherapeutically effective doses of the polypeptide of the invention toreduce the risk of developing cancers.

In vitro models can be used to determine the effective doses of thepolypeptide of the invention as a potential cancer treatment. These invitro models include proliferation assays of cultured tumor cells,growth of cultured tumor cells in soft agar (see Freshney, (1987)Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, NewYork, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described inGiovanella et al., J. Natl. Can. Inst., 52: 921-30 (1974), mobility andinvasive potential of tumor cells in Boyden Chamber assays as describedin Pilkington et al., Anticancer Res., 17: 4107-9 (1997), andangiogenesis assays such as induction of vascularization of the chickchorioallantoic membrane or induction of vascular endothelial cellmigration as described in Ribatta et al., Intl. J. Dev. Biol., 40:1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999)respectively. Suitable tumor cells lines are available, e.g. fromAmerican Type Tissue Culture Collection catalogs.

5.9.12 Receptor/Ligand Activity

A protein of the present invention may also demonstrate activity asreceptor, receptor ligand or inhibitor or agonist of receptor/ligandinteractions. A polynucleotide of the invention can encode a polypeptideexhibiting such characteristics. Examples of such receptors and ligandsinclude, without limitation, cytokine receptors and their ligands,receptor kinases and their ligands, receptor phosphatases and theirligands, receptors involved in cell-cell interactions and their ligands(including without limitation, cellular adhesion molecules (such asselectins, integrins and their ligands) and receptor/ligand pairsinvolved in antigen presentation, antigen recognition and development ofcellular and humoral immune responses. Receptors and ligands are alsouseful for screening of potential peptide or small molecule inhibitorsof the relevant receptor/ligand interaction. A protein of the presentinvention (including, without limitation, fragments of receptors andligands) may themselves be useful as inhibitors of receptor/ligandinteractions.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for receptor-ligand activity include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol.Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

By way of example, the polypeptides of the invention may be used as areceptor for a ligand(s) thereby transmitting the biological activity ofthat ligand(s). Ligands may be identified through binding assays,affinity chromatography, dihybrid screening assays, BIAcore assays, geloverlay assays, or other methods known in the art.

Studies characterizing drugs or proteins as agonist or antagonist orpartial agonists or a partial antagonist require the use of otherproteins as competing ligands. The polypeptides of the present inventionor ligand(s) thereof may be labeled by being coupled to radioisotopes,colorimetric molecules or a toxin molecules by conventional methods.(“Guide to Protein Purification”. Murray P. Deutscher (ed) Methods inEnzymology Vol. 182 (1990) Academic Press, Inc. San Diego). Examples ofradioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to,fluorescent molecules such as fluorescamine, or rhodamine or othercolorimetric molecules. Examples of toxins include, but are not limited,to ricin.

5.9.13 Drug Screening

This invention is particularly useful for screening chemical compoundsby using the novel polypeptides or binding fragments thereof in any of avariety of drug screening techniques. The polypeptides or fragmentsemployed in such a test may either be free in solution, affixed to asolid support, borne on a cell surface or located intracellularly. Onemethod of drug screening utilizes eukaryotic or prokaryotic host cellswhich are stably transformed with recombinant nucleic acids expressingthe polypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between polypeptides of theinvention or fragments and the agent being tested or examine thediminution in complex formation between the novel polypeptides and anappropriate cell line, which are well known in the art.

Sources for test compounds that may be screened for ability to bind toor modulate (i.e., increase or decrease) the activity of polypeptides ofthe invention include (1) inorganic and organic chemical libraries, (2)natural product libraries, and (3) combinatorial libraries comprised ofeither random or mimetic peptides, oligonucleotides or organicmolecules.

Chemical libraries may be readily synthesized or purchased from a numberof commercial sources, and may include structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” vianatural product screening.

The sources of natural product libraries are microorganisms (includingbacteria and fungi), animals, plants or other vegetation, or marineorganisms, and libraries of mixtures for screening may be created by:(1) fermentation and extraction of broths from soil, plant or marinemicroorganisms or (2) extraction of the organisms themselves. Naturalproduct libraries include polyketides, non-ribosomal peptides, and(non-naturally occurring) variants thereof. For a review, see Science282:63-68 (1998).

Combinatorial libraries are composed of large numbers of peptides,oligonucleotides or organic compounds and can be readily prepared bytraditional automated synthesis methods, PCR, cloning or proprietarysynthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include peptide, protein, peptidomimetic, multiparallelsynthetic collection, recombinatorial, and polypeptide libraries. For areview of combinatorial chemistry and libraries created therefrom, seeMyers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews andexamples of peptidomimetic libraries, see Al-Obeidi et al., Mol.Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996)(alkylated dipeptides).

Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to bind a polypeptide of theinvention. The molecules identified in the binding assay are then testedfor antagonist or agonist activity in in vivo tissue culture or animalmodels that are well known in the art. In brief, the molecules aretitrated into a plurality of cell cultures or animals and then testedfor either cell/animal death or prolonged survival of the animal/cells.

The binding molecules thus identified may be complexed with toxins,e.g., ricin or cholera, or with other compounds that are toxic to cellssuch as radioisotopes. The toxin-binding molecule complex is thentargeted to a tumor or other cell by the specificity of the bindingmolecule for a polypeptide of the invention. Alternatively, the bindingmolecules may be complexed with imaging agents for targeting and imagingpurposes.

5.9.14 Assay for Receptor Activity

The invention also provides methods to detect specific binding of apolypeptide e.g. a ligand or a receptor. The art provides numerousassays particularly useful for identifying previously unknown bindingpartners for receptor polypeptides of the invention. For example,expression cloning using mammalian or bacterial cells, or dihybridscreening assays can be used to identify polynucleotides encodingbinding partners. As another example, affinity chromatography with theappropriate immobilized polypeptide of the invention can be used toisolate polypeptides that recognize and bind polypeptides of theinvention. There are a number of different libraries used for theidentification of compounds, and in particular small molecule, thatmodulate (i.e., increase or decrease) biological activity of apolypeptide of the invention. Ligands for receptor polypeptides of theinvention can also be identified by adding exogenous ligands, orcocktails of ligands to two cells populations that are geneticallyidentical except for the expression of the receptor of the invention:one cell population expresses the receptor of the invention whereas theother does not. The response of the two cell populations to the additionof ligands(s) are then compared. Alternatively, an expression librarycan be co-expressed with the polypeptide of the invention in cells andassayed for an autocrine response to identify potential ligand(s). Asstill another example, BIAcore assays, gel overlay assays, or othermethods known in the art can be used to identify binding partnerpolypeptides, including, (1) organic and inorganic chemical libraries,(2) natural product libraries, and (3) combinatorial libraries comprisedof random peptides, oligonucleotides or organic molecules.

The role of downstream intracellular signaling molecules in thesignaling cascade of the polypeptide of the invention can be determined.For example, a chimeric protein in which the cytoplasmic domain of thepolypeptide of the invention is fused to the extracellular portion of aprotein, whose ligand has been identified, is produced in a host cell.The cell is then incubated with the ligand specific for theextracellular portion of the chimeric protein, thereby activating thechimeric receptor. Known downstream proteins involved in intracellularsignaling can then be assayed for expected modifications i.e.phosphorylation. Other methods known to those in the art can also beused to identify signaling molecules involved in receptor activity.

5.9.15 Anti-Inflammatory Activity

Compositions of the present invention may also exhibit anti-inflammatoryactivity. The anti-inflammatory activity may be achieved by providing astimulus to cells involved in the inflammatory response, by inhibitingor promoting cell-cell interactions (such as, for example, celladhesion), by inhibiting or promoting chemotaxis of cells involved inthe inflammatory process, inhibiting or promoting cell extravasation, orby stimulating or suppressing production of other factors which moredirectly inhibit or promote an inflammatory response. Compositions withsuch activities can be used to treat inflammatory conditions includingchronic or acute conditions), including without limitation intimationassociated with infection (such as septic shock, sepsis or systemicinflammatory response syndrome (SIRS)), ischemia-reperfusion injury,endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Compositions of theinvention may also be useful to treat anaphylaxis and hypersensitivityto an antigenic substance or material. Compositions of this inventionmay be utilized to prevent or treat condition such as, but not limitedto, utilized, for example, as part of methods for the prevention and/ortreatment of disorders involving sepsis, acute pancreatitis, endotoxinshock, cytokine induced shock, rheumatoid arthritis, chronicinflammatory arthritis, pancreatic cell damage from diabetes mellitustype 1, graft versus host disease, inflammatory bowel disease,inflamation associated with pulmonary disease, other autoimmune diseaseor inflammatory disease, an antiproliferative agent such as for acute orchronic mylegenous leukemia or in the prevention of premature laborsecondary to intrauterine infections.

5.9.16 Leukemias

Leukemias and related disorders may be treated or prevented byadministration of a therapeutic that promotes or inhibits function ofthe polynucleotides and/or polypeptides of the invention. Such leukemiasand related disorders include but are not limited to acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronicleukemia, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia (for a review of such disorders, see Fishman etal., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).

5.9.17 Nervous System Disorders

Nervous system disorders, involving cell types which can be tested forefficacy of intervention with compounds that modulate the activity ofthe polynucleotides and/or polypeptides of the invention, and which canbe treated upon thus observing an indication of therapeutic utility,include but are not limited to nervous system injuries, and diseases ordisorders which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated in a patient (including human and non-humanmammalian patients) according to the invention include but are notlimited to the following lesions of either the central (including spinalcord, brain) or peripheral nervous systems:

-   -   (i) traumatic lesions, including lesions caused by physical        injury or associated with surgery, for example, lesions which        sever a portion of the nervous system, or compression injuries;    -   (ii) ischemic lesions, in which a lack of oxygen in a portion of        the nervous system results in neuronal injury or death,        including cerebral infarction or ischemia, or spinal cord        infarction or ischemia;    -   (iii) infectious lesions, in which a portion of the nervous        system is destroyed or injured as a result of infection, for        example, by an abscess or associated with infection by human        immunodeficiency virus, herpes zoster, or herpes simplex virus        or with Lyme disease, tuberculosis, syphilis;    -   (iv) degenerative lesions, in which a portion of the nervous        system is destroyed or injured as a result of a degenerative        process including but not limited to degeneration associated        with Parkinson's disease, Alzheimer's disease, Huntington's        chorea, or amyotrophic lateral sclerosis;    -   (v) lesions associated with nutritional diseases or disorders,        in which a portion of the nervous system is destroyed or injured        by a nutritional disorder or disorder of metabolism including        but not limited to, vitamin B12 deficiency, folic acid        deficiency, Wernicke disease, tobacco-alcohol amblyopia,        Marchiafava-Bignami disease (primary degeneration of the corpus        callosum), and alcoholic cerebellar degeneration;    -   (vi) neurological lesions associated with systemic diseases        including but not limited to diabetes (diabetic neuropathy,        Bell's palsy), systemic lupus erythematosus, carcinoma, or        sarcoidosis;    -   (vii) lesions caused by toxic substances including alcohol,        lead, or particular neurotoxins; and    -   (viii) demyelinated lesions in which a portion of the nervous        system is destroyed or injured by a demyelinating disease        including but not limited to multiple sclerosis, human        immunodeficiency virus-associated myelopathy, transverse        myelopathy or various etiologies, progressive multifocal        leukoencephalopathy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatmentof a nervous system disorder may be selected by testing for biologicalactivity in promoting the survival or differentiation of neurons. Forexample, and not by way of limitation, therapeutics which elicit any ofthe following effects may be useful according to the invention:

-   -   (i) increased survival time of neurons in culture;    -   (ii) increased sprouting of neurons in culture or in vivo;    -   (iii) increased production of a neuron-associated molecule in        culture or in vivo, e.g., choline acetyltransferase or        acetylcholinesterase with respect to motor neurons; or    -   (iv) decreased symptoms of neuron dysfunction in vivo.

Such effects may be measured by any method known in the art. Inpreferred, non-limiting embodiments, increased survival of neurons maybe measured by the method set forth in Arakawa et al. (1990, J.Neurosci. 10:3507-3515); increased sprouting of neurons may be detectedby methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) orBrown et al. (1981, Ann. Rev. Neurosci. 4:1742); increased production ofneuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., depending on themolecule to be measured; and motor neuron dysfunction may be measured byassessing the physical manifestation of motor neuron disorder, e.g.,weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treatedaccording to the invention include but are not limited to disorders suchas infarction, infection, exposure to toxin, trauma, surgical damage,degenerative disease or malignancy that may affect motor neurons as wellas other components of the nervous system, as well as disorders thatselectively affect neurons such as amyotrophic lateral sclerosis, andincluding but not limited to progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

5.9.18 Arthritis and Inflammation

The immunosuppressive effects of the compositions of the inventionagainst rheumatoid arthritis is determined in an experimental animalmodel system. The experimental model system is adjuvant inducedarthritis in rats, and the protocol is described by J. Holoshitz, etat., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int. Arch.Allergy Appl. Immunol., 23:129. Induction of the disease can be causedby a single injection, generally intradermally, of a suspension ofkilled Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).The route of injection can vary, but rats may be injected at the base ofthe tail with an adjuvant mixture. The inhibitor is administered inphosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. Thecontrol consists of administering PBS only.

The procedure for testing the effects of the test compound would consistof intradermally injecting killed Mycobacterium tuberculosis in CFAfollowed by immediately administering the inhibitor and subsequenttreatment every other day until day 24. At 14, 15, 18, 20, 22, and 24days after injection of Mycobacterium CFA, an overall arthritis scoremay be obtained as described by J. Holoskitz above. An analysis of thedata would reveal that the test compound would have a dramatic affect onthe swelling of the joints as measured by a decrease of the arthritisscore.

5.9.19 Other Activities

A protein of the invention may also exhibit one or more of the followingadditional activities or effects: inhibiting the growth, infection orfunction of, or killing, infectious agents, including, withoutlimitation, bacteria, viruses, fungi and other parasites; effecting(suppressing or enhancing) bodily characteristics, including, withoutlimitation, height, weight, hair color, eye color, skin, fat to leanratio or other tissue pigmentation, or organ or body part size or shape(such as, for example, breast augmentation or diminution, change in boneform or shape); effecting biorhythms or circadian cycles or rhythms;effecting the fertility of male or female subjects; effecting themetabolism, catabolism, anabolism, processing, utilization, storage orelimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, co-factors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

5.9.20 Identification of Polymorphisms

The demonstration of polymorphisms makes possible the identification ofsuch polymorphisms in human subjects and the pharmacogenetic use of thisinformation for diagnosis and treatment. Such polymorphisms may beassociated with, e.g., differential predisposition or susceptibility tovarious disease states (such as disorders involving inflammation orimmune response) or a differential response to drug administration, andthis genetic information can be used to tailor preventive or therapeutictreatment appropriately. For example, the existence of a polymorphismassociated with a predisposition to inflammation or autoimmune diseasemakes possible the diagnosis of this condition in humans by identifyingthe presence of the polymorphism.

Polymorphisms can be identified in a variety of ways known in the artwhich all generally involve obtaining a sample from a patient, analyzingDNA from the sample, optionally involving isolation or amplification ofthe DNA, and identifying the presence of the polymorphism in the DNA.For example, PCR may be used to amplify an appropriate fragment ofgenomic DNA which may then be sequenced. Alternatively, the DNA may besubjected to allele-specific oligonucleotide hybridization (in whichappropriate oligonucleotides are hybridized to the DNA under conditionspermitting detection of a single base mismatch) or to a singlenucleotide extension assay (in which an oligonucleotide that hybridizesimmediately adjacent to the position of the polymorphism is extendedwith one or more labeled nucleotides). In addition, traditionalrestriction fragment length polymorphism analysis (using restrictionenzymes that provide differential digestion of the genomic DNA dependingon the presence or absence of the polymorphism) may be performed. Arrayswith nucleotide sequences of the present invention can be used to detectpolymorphisms. The array can comprise modified nucleotide sequences ofthe present invention in order to detect the nucleotide sequences of thepresent invention. In the alternative, any one of the nucleotidesequences of the present invention can be placed on the array to detectchanges from those sequences.

Alternatively a polymorphism resulting in a change in the amino acidsequence could also be detected by detecting a corresponding change inamino acid sequence of the protein, e.g., by an antibody specific to thevariant sequence.

5.10 Therapeutic Methods

The compositions (including polypeptide fragments, analogs, variants andantibodies or other binding partners or modulators including antisensepolynucleotides) of the invention have numerous applications in avariety of therapeutic methods. Examples of therapeutic applicationsinclude, but are not limited to, those exemplified herein.

5.10.1 Examples

Another embodiment of the invention is the administration of aneffective amount of the polypeptide or other composition of theinvention to individuals affected by a disease or disorder which can bemodulated by regulating the IgSF member of the invention. While the modeof administration is not particularly important, parenteraladministration is preferred. An exemplary mode of administration is todeliver an intravenous bolus. The dosage of the polypeptide orcomposition of the invention will normally be determined by theprescribing physician. It is to be expected that the dosage will varyaccording to the age, weight, condition and response of the individualpatient. Typically, the amount of protein or other active ingredientadministered per dose will be in the range of about 0.1 to 25 mg/kg ofbody weight, with the preferred dose being about 0.1 to 10 mg/kg ofpatient body weight. For parenteral administration, the polypeptides orother active ingredient of the invention will be formulated in aninjectable form that includes a pharmaceutically acceptable parenteralvehicle. Such vehicles are well known in the art and examples includewater, saline, Ringer's solution, dextrose solution, and solutionsconsisting of small amounts of the human serum albumin. The vehicle maycontain minor amounts of additives that maintain the isotonicity andstability of the polypeptide or other active ingredient. The preparationof such solutions is within the skill of the art. Typically, thecytokine inhibitor will be formulated in such vehicles at aconcentration of about 1-8 mg/ml to about 10 mg/ml.

5.11 Pharmaceutical Formulations and Routes of Administration

A protein or other composition of the present invention (from whateversource derived, including without limitation from recombinant andnon-recombinant sources and including antibodies and other bindingpartners of the polypeptides of the invention) may be administered to apatient in need, by itself, or in pharmaceutical compositions where itis mixed with suitable carriers or excipient(s) at doses to treat orameliorate a variety of disorders. Such a composition may optionallycontain (in addition to protein or other active ingredient and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF-, TNF1, TNF2, G-CSF,Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. Infurther compositions, proteins of the invention may be combined withother agents beneficial to the treatment of the bone and/or cartilagedefect, wound, or tissue in questions. These agents include variousgrowth factors such as epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), transforming growth factors (TGF- and TGF-),insulin-like growth factor (IGF), as well as cytokines described herein.

The pharmaceutical composition may further contain other agents whicheither enhance the activity of the protein or other active ingredient orcompliment its activity or use in treatment. Such additional factorsand/or agents may be included in the pharmaceutical composition toproduce a synergistic effect with protein or other active ingredient ofthe invention, or to minimize side effects. Conversely, protein or otheractive ingredient of the present invention may be included informulations of the particular cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent to minimize side effects of the cytokine, lymphokine, otherhematopoietic factor, thrombolytic or anti-thrombotic factor, oranti-inflammatory agent. A protein of the present invention may beactive in multimers (e.g., heterodimers or homodimers) or complexes withitself or other proteins. As a result, pharmaceutical compositions ofthe invention may comprise a protein of the invention in such multimericor complexed form.

As an alternative to being included in a pharmaceutical composition ofthe invention including a first protein, a second protein or atherapeutic agent may be concurrently administered with the firstprotein (e.g., at the same time, or at differing times provided thattherapeutic concentrations of the combination of agents is achieved atthe treatment site). Techniques for formulation and administration ofthe compounds of the instant application may be found in “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latestedition. A therapeutically effective dose further refers to that amountof the compound sufficient to result in amelioration of symptoms, e.g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein or other active ingredientof the present invention is administered to a mammal having a conditionto be treated. Protein or other active ingredient of the presentinvention may be administered in accordance with the method of theinvention either alone or in combination with other therapies such astreatments employing cytokines, lymphokines or other hematopoieticfactors. When co-administered with one or more cytokines, lymphokines orother hematopoietic factors, protein or other active ingredient of thepresent invention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein or other active ingredient of thepresent invention in combination with cytokine(s), lymphokine(s), otherhematopoietic factor(s), thrombolytic or anti-thrombotic factors.

5.11.1 Routes of Administration

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof protein or other active ingredient of the present invention used inthe pharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal, parenteral or intravenous injection.Intravenous administration to the patient is preferred.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a arthritic joints or in fibrotic tissue, often in a depot orsustained release formulation. In order to prevent the scarring processfrequently occurring as complication of glaucoma surgery, the compoundsmay be administered topically, for example, as eye drops. Furthermore,one may administer the drug in a targeted drug delivery system, forexample, in a liposome coated with a specific antibody, targeting, forexample, arthritic or fibrotic tissue. The liposomes will be targeted toand taken up selectively by the afflicted tissue.

The polypeptides of the invention are administered by any route thatdelivers an effective dosage to the desired site of action. Thedetermination of a suitable route of administration and an effectivedosage for a particular indication is within the level of skill in theart. Preferably for wound treatment, one administers the therapeuticcompound directly to the site. Suitable dosage ranges for thepolypeptides of the invention can be extrapolated from these dosages orfrom similar studies in appropriate animal models. Dosages can then beadjusted as necessary by the clinician to provide maximal therapeuticbenefit.

5.11.2 Compositions/Formulations

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in a conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known, e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein or other active ingredient of the present invention isadministered orally, protein or other active ingredient of the presentinvention will be in the form of a tablet, capsule, powder, solution orelixir. When administered in tablet form, the pharmaceutical compositionof the invention may additionally contain a solid carrier such as agelatin or an adjuvant. The tablet, capsule, and powder contain fromabout 5 to 95% protein or other active ingredient of the presentinvention, and preferably from about 25 to 90% protein or other activeingredient of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein or other active ingredient of the present invention,and preferably from about 1 to 50% protein or other active ingredient ofthe present invention.

When a therapeutically effective amount of protein or other activeingredient of the present invention is administered by intravenous,cutaneous or subcutaneous injection, protein or other active ingredientof the present invention will be in the form of a pyrogen-free,parenterally acceptable aqueous solution. The preparation of suchparenterally acceptable protein or other active ingredient solutions,having due regard to pH, isotonicity, stability, and the like, is withinthe skill in the art. A preferred pharmaceutical composition forintravenous, cutaneous, or subcutaneous injection should contain, inaddition to protein or other active ingredient of the present invention,an isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thepharmaceutical composition of the present invention may also containstabilizers, preservatives, buffers, antioxidants, or other additivesknown to those of skill in the art. For injection, the agents of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose. Alternatively, otherdelivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Varioustypes of sustained-release materials have been established and are wellknown by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the compounds for a fewweeks up to over 100 days. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein or other active ingredient stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols. Many of the active ingredients of theinvention may be provided as salts with pharmaceutically compatiblecounterions. Such pharmaceutically acceptable base addition salts arethose salts which retain the biological effectiveness and properties ofthe free acids and which are obtained by reaction with inorganic ororganic bases such as sodium hydroxide, magnesium hydroxide, ammonia,trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodiumacetate, potassium benzoate, triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) or other active ingredient of presentinvention along with protein or peptide antigens. The protein and/orpeptide antigen will deliver a stimulatory signal to both B and Tlymphocytes. B lymphocytes will respond to antigen through their surfaceimmunoglobulin receptor. T Lymphocytes will respond to antigen throughthe T cell receptor (TCR) following presentation of the antigen by MHCproteins. MHC and structurally related proteins including those encodedby class I and class II MHC genes on host cells will serve to presentthe peptide antigen(s) to T lymphocytes. The antigen components couldalso be supplied as purified MHC-peptide complexes alone or withco-stimulatory molecules that can directly signal T cells. Alternativelyantibodies able to bind surface immunoglobulin and other molecules on Bcells as well as antibodies able to bind the TCR and other molecules onT cells can be combined with the pharmaceutical composition of theinvention. The pharmaceutical composition of the invention may be in theform of a liposome in which protein of the present invention iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides, sulfatides,lysolecithins, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871;4,501,728; 4,837,028; and 4,737,323, all of which are incorporatedherein by reference.

The amount of protein or other active ingredient of the presentinvention in the pharmaceutical composition of the present inventionwill depend upon the nature and severity of the condition being treated,and on the nature of prior treatments which the patient has undergone.Ultimately, the attending physician will decide the amount of protein orother active ingredient of the present invention with which to treateach individual patient. Initially, the attending physician willadminister low doses of protein or other active ingredient of thepresent invention and observe the patient's response. Larger doses ofprotein or other active ingredient of the present invention may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. It iscontemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 0.01μg to about 100 mg (preferably about 0.1 μg to about 10 mg, morepreferably about 0.1 μg to about 1 mg) of protein or other activeingredient of the present invention per kg body weight. For compositionsof the present invention which are useful for bone, cartilage, tendon orligament regeneration, the therapeutic method includes administering thecomposition topically, systematically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of bone, cartilage or tissuedamage. Topical administration may be suitable for wound healing andtissue repair. Therapeutically useful agents other than a protein orother active ingredient of the invention which may also optionally beincluded in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. Preferably for bone and/orcartilage formation, the composition would include a matrix capable ofdelivering the protein-containing or other active ingredient-containingcomposition to the site of bone and/or cartilage damage, providing astructure for the developing bone and cartilage and optimally capable ofbeing resorbed into the body. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability. Presentlypreferred is a 50:50 (mole weight) copolymer of lactic acid and glycolicacid in the form of porous particles having diameters ranging from 150to 800 microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorption of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins orother active ingredient of the invention may be combined with otheragents beneficial to the treatment of the bone and/or cartilage defect,wound, or tissue in question. These agents include various growthfactors such as epidermal growth factor (EGF), platelet derived growthfactor (PDGF), transforming growth factors (TGF- and TGF-), andinsulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins or other active ingredient of the present invention. The dosageregimen of a protein-containing pharmaceutical composition to be used intissue regeneration will be determined by the attending physicianconsidering various factors which modify the action of the proteins,e.g., amount of tissue weight desired to be formed, the site of damage,the condition of the damaged tissue, the size of a wound, type ofdamaged tissue (e.g., bone), the patient's age, sex, and diet, theseverity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

5.11.3. Effective Dosage

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein. For any compound used in the methodof the invention, the therapeutically effective dose can be estimatedinitially from appropriate in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat can be used to more accurately determine useful doses in humans.For example, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture (i.e., the concentration of the test compound whichachieves a half-maximal inhibition of the IgSF protein's bio logicalactivity). Such information can be used to more accurately determineuseful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds which exhibit high therapeutic indicesare preferred. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage of such compounds lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p.1. Dosage amount and interval may be adjusted individually toprovide plasma levels of the active moiety which are sufficient tomaintain the desired effects, or minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from in vitrodata. Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%. In cases of local administration or selectiveuptake, the effective local concentration of the drug may not be relatedto plasma concentration.

An exemplary dosage regimen for polypeptides or other compositions ofthe invention will be in the range of about 0.01 to 100 mg/kg of bodyweight daily, with the preferred dose being about 0.1 to 25 mg/kg ofpatient body weight daily, varying in adults and children. Dosing may beonce daily, or equivalent doses may be delivered at longer or shorterintervals.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's age and weight, the severityof the affliction, the manner of administration and the judgment of theprescribing physician.

5.11.4. Packaging

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga compound of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition.

5.12 Antibodies

Also included in the invention are antibodies to proteins, or fragmentsof proteins of the invention. The term “antibody” as used herein refersto immunoglobulin molecules and immunologically active portions ofimmunoglobulin (Ig) molecules, i.e., molecules that contain anantigen-binding site that specifically binds (immunoreacts with) anantigen. Such antibodies include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, F_(ab), F_(ab′) and F_((ab′)2)fragments, and an F_(ab) expression library. In general, an antibodymolecule obtained from humans relates to any of the classes IgG, IgM,IgA, IgE, and IgD, which differ from one another by the nature of theheavy chain present in the molecule. Certain classes have subclasses aswell, such as IgG₁, IgG₂, and others. Furthermore, in humans, the lightchain may be a kappa chain or a lambda chain. Reference herein toantibodies includes a reference to all such classes, subclasses, andtypes of human antibody species.

An isolated related protein of the invention may be intended to serve asan antigen, or a portion or fragment thereof, and additionally can beused as an immunogen to generate antibodies that immunospecifically bindthe antigen, using standard techniques for polyclonal and monoclonalantibody preparation. The full-length protein can be used or,alternatively, the invention provides antigenic peptide fragments of theantigen for use as immunogens. An antigenic peptide fragment comprisesat least 6 amino acid residues of the amino acid sequence of the fulllength protein, such as an amino acid sequence shown in SEQ ID NO: 10,13-24, 32 or 34 and encompasses an epitope thereof such that an antibodyraised against the peptide forms a specific immune complex with the fulllength protein or with any fragment that contains the epitope.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, or at least 15 amino acid residues, or at least 20 amino acidresidues, or at least 30 amino acid residues. Preferred epitopesencompassed by the antigenic peptide are regions of the protein that arelocated on its surface; commonly these are hydrophilic regions.

In certain embodiments of the invention, at least one epitopeencompassed by the antigenic peptide is a region of TGF alpha-likeprotein that is located on the surface of the protein, e.g., ahydrophilic region. A hydrophobicity analysis of the human relatedprotein sequence will indicate which regions of a related protein areparticularly hydrophilic and, therefore, are likely to encode surfaceresidues useful for targeting antibody production. As a means fortargeting antibody production, hydropathy plots showing regions ofhydrophilicity and hydrophobicity may be generated by any method wellknown in the art, including, for example, the Kyte Doolittle or the HoppWoods methods, either with or without Fourier transformation. See, e.g.,Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte andDoolittle 1982, J. Mol. Biol. 157: 105-142, each of which isincorporated herein by reference in its entirety. Antibodies that arespecific for one or more domains within an antigenic protein, orderivatives, fragments, analogs or homologs thereof, are also providedherein.

A protein of the invention, or a derivative, fragment, analog, homologor ortholog thereof, may be utilized as an immunogen in the generationof antibodies that immunospecifically bind these protein components.

The term “specific for” indicates that the variable regions of theantibodies of the invention recognize and bind polypeptides of theinvention exclusively (i.e., able to distinguish the polypeptide of theinvention from other similar polypeptides despite sequence identity,homology, or similarity found in the family of polypeptides), but mayalso interact with other proteins (for example, S. aureus protein A orother antibodies in ELISA techniques) through interactions withsequences outside the variable region of the antibodies, and inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody of the invention are wellknown and routinely practiced in the art. For a comprehensive discussionof such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual;Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter6. Antibodies that recognize and bind fragments of the polypeptides ofthe invention are also contemplated, provided that the antibodies arefirst and foremost specific for, as defined above, full-lengthpolypeptides of the invention. As with antibodies that are specific forfull length polypeptides of the invention, antibodies of the inventionthat recognize fragments are those which can distinguish polypeptidesfrom the same family of polypeptides despite inherent sequence identity,homology, or similarity found in the family of proteins.

Antibodies of the invention are useful for, for example, therapeuticpurposes (by modulating activity of a polypeptide of the invention),diagnostic purposes to detect or quantitate a polypeptide of theinvention, as well as purification of a polypeptide of the invention.Kits comprising an antibody of the invention for any of the purposesdescribed herein are also comprehended. In general, a kit of theinvention also includes a control antigen for which the antibody isimmunospecific. The invention further provides a hybridoma that producesan antibody according to the invention. Antibodies of the invention areuseful for detection and/or purification of the polypeptides of theinvention.

Monoclonal antibodies binding to the protein of the invention may beuseful diagnostic agents for the immunodetection of the protein.Neutralizing monoclonal antibodies binding to the protein may also beuseful therapeutics for both conditions associated with the protein andalso in the treatment of some forms of cancer where abnormal expressionof the protein is involved. In the case of cancerous cells or leukemiccells, neutralizing monoclonal antibodies against the protein may beuseful in detecting and preventing the metastatic spread of thecancerous cells, which may be mediated by the protein.

The labeled antibodies of the present invention can be used for invitro, in vivo, and in situ assays to identify cells or tissues in whicha fragment of the polypeptide of interest is expressed. The antibodiesmay also be used directly in therapies or other diagnostics. The presentinvention further provides the above-described antibodies immobilized ona solid support. Examples of such solid supports include plastics suchas polycarbonate, complex carbohydrates such as agarose and Sepharose®,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed.,.Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986);Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). Theimmobilized antibodies of the present invention can be used for invitro, in vivo, and in situ assays as well as for immuno-affinitypurification of the proteins of the present invention.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs, ororthologs thereof (see, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference). Some of theseantibodies are discussed below.

5.12.1 Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byone or more injections with the native protein, a synthetic variantthereof, or a derivative of the foregoing. An appropriate immunogenicpreparation can contain, for example, the naturally occurringimmunogenic protein, a chemically synthesized polypeptide representingthe immunogenic protein, or a recombinantly expressed immunogenicprotein. Furthermore, the protein may be conjugated to a second proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. The preparation can further include an adjuvant. Variousadjuvants used to increase the immunological response include, but arenot limited to, Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface-active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,etc.), adjuvants usable in humans such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory agents. Additionalexamples of adjuvants that can be employed include MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomy colate).

The polyclonal antibody molecules directed against the immunogenicprotein can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

5.12.2 Monoclonal Antibodies

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen-binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof, or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine, and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having ahigh degree of specificity and a high binding affinity for the targetantigen are isolated.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include, for example, Dulbecco'sModified Eagle's Medium and RPMI-1640 medium. Alternatively, thehybridoma cells can be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

5.12.3 Humanized Antibodies

The antibodies directed against the protein antigens of the inventioncan further comprise humanized antibodies or human antibodies. Theseantibodies are suitable for administration to humans without engenderingan immune response by the human against the administered immunoglobulin.Humanized forms of antibodies are chimeric immunoglobulins,immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) that areprincipally comprised of the sequence of a human immunoglobulin, andcontain minimal sequence derived from a non-human immunoglobulin.Humanization can be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539). In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues that are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

5.12.4 Human Antibodies

Fully human antibodies relate to antibody molecules in which essentiallythe entire sequences of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies”, or “fully human antibodies “herein. Human monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) andthe EBV hybridoma technique to produce human monoclonal antibodies (seeCole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized inthe practice of the present invention and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859(1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (NatureBiotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14,826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93(1995)).

Human antibodies may additionally be produced using transgenic nonhumananimals that are modified so as to produce fully human antibodies ratherthan the animal's endogenous antibodies in response to challenge by anantigen. (See PCT publication WO94/02602). The endogenous genes encodingthe heavy and light immunoglobulin chains in the nonhuman host have beenincapacitated, and active loci encoding human heavy and light chainimmunoglobulins are inserted into the host's genome. The hu man genesare incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells that secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a methodincluding deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene-encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

5.12.5 FAB Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to an antigenic protein of theinvention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods canbe adapted for the construction of F_(ab) expression libraries (seee.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid andeffective identification of monoclonal F_(ab) fragments with the desiredspecificity for a protein or derivatives, fragments, analogs or homologsthereof. Antibody fragments that contain the idiotypes to a proteinantigen may be produced by techniques known in the art including, butnot limited to: (i) an F_((ab′)2) fragment produced by pepsin digestionof an antibody molecule; (ii) an F_(ab) fragment generated by reducingthe disulfide bridges of an F_((ab′)2) fragment; (iii) an F_(ab)fragment generated by the treatment of the antibody molecule with papainand a reducing agent and (iv) F_(v) fragments.

5.12.6 Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is foran antigenic protein of the invention. The second binding target is anyother antigen, and advantageously is a cell-surface protein or receptoror receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., 1991 EMBO J.,10:3655-3659.

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers that are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64), Fc RII (CD32)and Fc RIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

5.12.7 Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies can be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

5.12.8 Effector Function Engineering

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

5.12.9 Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See. WO94/11026.

In another embodiment, the antibody can be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is in turnconjugated to a cytotoxic agent.

5.13 Computer Readable Sequences

In one application of this embodiment, a nucleotide sequence of thepresent invention can be recorded on computer readable media. As usedherein, “computer readable media” refers to any medium which can be readand accessed directly by a computer. Such media include, but are notlimited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention. As used herein, recorded” refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide sequence of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the nucleotide sequenceinformation of the present invention on computer readable medium. Thesequence information can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMicrosoft Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of data processorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the nucleotide sequenceinformation of the present invention.

By providing any of the nucleotide sequences SEQ ID NO: 1-9, 11, 12, 31or 33 or a representative fragment thereof; or a nucleotide sequence atleast 99.9% identical to any of the nucleotide sequences of the SEQ IDNO: 1-9, 11, 12, 31 or 33 in computer readable form, a skilled artisancan routinely access the sequence information for a variety of purposes.Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable medium.The examples which follow demonstrate how software which implements theBLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE(Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on aSybase system is used to identify open reading frames (ORFs) within anucleic acid sequence. Such ORFs may be protein encoding fragments andmay be useful in producing commercially important proteins such asenzymes used in fermentation reactions and in the production ofcommercially useful metabolites.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the nucleotidesequence information of the present invention. The minimum hardwaremeans of the computer-based systems of the present invention comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems are suitable for use inthe present invention. As stated above, the computer-based systems ofthe present invention comprise a data storage means having storedtherein a nucleotide sequence of the present invention and the necessaryhardware means and software means for supporting and implementing asearch means. As used herein, “data storage means” refers to memorywhich can store nucleotide sequence information of the presentinvention, or a memory access means which can access manufactures havingrecorded thereon the nucleotide sequence information of the presentinvention.

As used herein, “search means” refers to one or more programs which areimplemented on the computer-based system to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of a known sequence which match a particular target sequence ortarget motif. A variety of known algorithms are disclosed publicly and avariety of commercially available software for conducting search meansare and can be used in the computer-based systems of the presentinvention. Examples of such software includes, but is not limited to,Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). Askilled artisan can readily recognize that any one of the availablealgorithms or implementing software packages for conducting homologysearches can be adapted for use in the present computer-based systems.As used herein, a “target sequence” can be any nucleic acid or aminoacid sequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized that searchesfor commercially important fragments, such as sequence fragmentsinvolved in gene expression and protein processing, may be of shorterlength.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

5.14 Expression Modulating Sequences

EMF sequences can be identified within a genome by their proximity tothe ORFs. An intergenic segment, or a fragment of the intergenicsegment, from about 10 to 200 nucleotides in length, taken 5′ from anyORF will modulate the expression of an operably linked 3′ ORF in afashion similar to that found with the naturally linked ORF sequence. Asused herein, an “intergenic segment” refers to the fragments of a genomewhich are between two ORF(S) herein described. Alternatively, EMFs canbe identified using known EMFs as a target sequence or target motif inthe computer-based systems of the present invention.

The presence and activity of an EMF can be confirmed using an EMF trapvector. An EMF trap vector contains a cloning site 5′ to a markersequence. A marker sequence encodes an identifiable phenotype, such asantibiotic resistance or a complementing nutrition auxotrophic factor,which can be identified or assayed when the EMF trap vector is placedwithin an appropriate host under appropriate conditions. As describedabove, an EMF will modulate the expression of an operably linked markersequence. A more detailed discussion of various marker sequences isprovided below. A sequence which is suspected of being an EMF is clonedin all three reading frames in one or more restriction sites upstreamfrom the marker sequence in the EMF trap vector. The vector is thentransformed into an appropriate host using known procedures and thephenotype of the transformed host is examined under appropriateconditions. As described above, an EMF will modulate the expression ofan operably linked marker sequence.

5.15 Triple Helix Formation

In addition, the fragments of the present invention, as broadlydescribed, can be used to control gene expression through triple helixformation or antisense DNA or RNA, both of which methods are based onthe binding of a polynucleotide sequence to DNA or RNA. Polynucleotidessuitable for use in these methods are usually 20 to 40 bases in lengthand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 15241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide.

5.16 Diagnostic Assays and Kits

The present invention further provides methods to identify the presenceor expression of one of the ORFs of the present invention, or homologthereof, in a test sample, using a nucleic acid probe or antibodies ofthe present invention, optionally conjugated or otherwise associatedwith a suitable label.

In general, methods for detecting a polynucleotide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polynucleotide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolynucleotide of the invention is detected in the sample. Such methodscan also comprise contacting a sample under stringent hybridizationconditions with nucleic acid primers that anneal to a polynucleotide ofthe invention under such conditions, and amplifying annealedpolynucleotides, so that if a polynucleotide is amplified, apolynucleotide of the invention is detected in the sample.

In general, methods for detecting a polypeptide of the invention cancomprise contacting a sample with a compound that binds to and forms acomplex with the polypeptide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolypeptide of the invention is detected in the sample.

In detail, such methods comprise incubating a test sample with one ormore of the antibodies or one or more of the nucleic acid probes of thepresent invention and assaying for binding of the nucleic acid probes orantibodies to components within the test sample.

Conditions for incubating a nucleic acid probe or antibody with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid probe or antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available hybridization,amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard, T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985). The test samplesof the present invention include cells, protein or membrane extracts ofcells, or biological fluids such as sputum, blood, serum, plasma, orurine. The test sample used in the above-described method will varybased on the assay format, nature of the detection method and thetissues, cells or extracts used as the sample to be assayed. Methods forpreparing protein extracts or membrane extracts of cells are well knownin the art and can be readily be adapted in order to obtain a samplewhich is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention. Specifically, the invention provides a compartment kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the probes or antibodies of thepresent invention; and (b) one or more other containers comprising oneor more of the following: wash reagents, reagents capable of detectingpresence of a bound probe or antibody.

In detail, a compartment kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe. Types of detectionreagents include labeled nucleic acid probes, labeled secondaryantibodies, or in the alternative, if the primary antibody is labeled,the enzymatic, or antibody binding reagents which are capable ofreacting with the labeled antibody. One skilled in the art will readilyrecognize that the disclosed probes and antibodies of the presentinvention can be readily incorporated into one of the established kitformats which are well known in the art.

5.17 Medical Imaging

The novel polypeptides and binding partners of the invention are usefulin medical imaging of sites expressing the molecules of the invention(e.g., where the polypeptide of the invention is involved in the immuneresponse, for imaging sites of inflammation or infection). See, e.g.,Kunkel et al., U.S. Pat. No. 5,413,778. Such methods involve chemicalattachment of a labeling or imaging agent, administration of the labeledpolypeptide to a subject in a pharmaceutically acceptable carrier, andimaging the labeled polypeptide in vivo at the target site.

5.18 Screening Assays

Using the isolated proteins and polynucleotides of the invention, thepresent invention further provides methods of obtaining and identifyingagents which bind to a polypeptide encoded by an ORF corresponding toany of the nucleotide sequences set forth in the SEQ ID NO: 1-9, 11, 12,31 or 33 or bind to a specific domain of the polypeptide encoded by thenucleic acid. In detail, said method comprises the steps of:

-   -   (a) contacting an agent with an isolated protein encoded by an        ORF of the present invention, or nucleic acid of the invention;        and    -   (b) determining whether the agent binds to said protein or said        nucleic acid.

In general, therefore, such methods for identifying compounds that bindto a polynucleotide of the invention can comprise contacting a compoundwith a polynucleotide of the invention for a time sufficient to form apolynucleotide/compound complex, and detecting the complex, so that if apolynucleotide/compound complex is detected, a compound that binds to apolynucleotide of the invention is identified.

Likewise, in general, therefore, such methods for identifying compoundsthat bind to a polypeptide of the invention can comprise contacting acompound with a polypeptide of the invention for a time sufficient toform a polypeptide/compound complex, and detecting the complex, so thatif a polypeptide/compound complex is detected, a compound that binds toa polynucleotide of the invention is identified.

Methods for identifying compounds that bind to a polypeptide of theinvention can also comprise contacting a compound with a polypeptide ofthe invention in a cell for a time sufficient to form apolypeptide/compound complex, wherein the complex drives expression of areceptor gene sequence in the cell, and detecting the complex bydetecting reporter gene sequence expression, so that if apolypeptide/compound complex is detected, a compound that binds apolypeptide of the invention is identified.

Compounds identified via such methods can include compounds whichmodulate the activity of a polypeptide of the invention (that is,increase or decrease its activity, relative to activity observed in theabsence of the compound). Alternatively, compounds identified via suchmethods can include compounds which modulate the expression of apolynucleotide of the invention (that is, increase or decreaseexpression relative to expression levels observed in the absence of thecompound). Compounds, such as compounds identified via the methods ofthe invention, can be tested using standard assays well known to thoseof skill in the art for their ability to modulate activity/expression.

The agents screened in the above assay can be, but are not limited to,peptides, carbohydrates, vitamin derivatives, or other pharmaceuticalagents. The agents can be selected and screened at random or rationallyselected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention. Alternatively, agents may be rationally selectedor designed. As used herein, an agent is said to be “rationally selectedor designed” when the agent is chosen based on the configuration of theparticular protein. For example, one skilled in the art can readilyadapt currently available procedures to generate peptides,pharmaceutical agents and the like capable of binding to a specificpeptide sequence in order to generate rationally designed antipeptidepeptides, for example see Hurby et al., Application of SyntheticPeptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide,W.H. Freeman, N.Y. (1992), pp. 289-307, and Kaspczak et al.,Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control. One class of DNA binding agents areagents which contain base residues which hybridize or form a triplehelix formation by binding to DNA or RNA. Such agents can be based onthe classic phosphodiester, ribonucleic acid backbone, or can be avariety of sulfhydryl or polymeric derivatives which have baseattachment capacity.

Agents suitable for use in these methods usually contain 20 to 40 basesand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide and other DNA binding agents. Agents whichbind to a protein encoded by one of the ORFs of the present inventioncan be used as a diagnostic agent, in the control of bacterial infectionby modulating the activity of the protein encoded by the ORF. Agentswhich bind to a protein encoded by one of the ORFs of the presentinvention can be formulated using known techniques to generate apharmaceutical composition.

5.19 Use of Nucleic Acids as Probes

Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from any ofthe nucleotide sequences SEQ ID NO: 1-9, 11, 12, 31 or 33. Because thecorresponding gene is only expressed in a limited number of tissues, ahybridization probe derived from of any of the nucleotide sequences SEQID NO: 1-9, 11, 12, 31 or 33 can be used as an indicator of the presenceof RNA of cell type of such a tissue in a sample.

Any suitable hybridization technique can be employed, such as, forexample, in situ hybridization. PCR as described in U.S. Pat. Nos.4,683,195 and 4,965,188 provides additional uses for oligonucleotidesbased upon the nucleotide sequences. Such probes used in PCR may be ofrecombinant origin, may be chemically synthesized, or a mixture of both.The probe will comprise a discrete nucleotide sequence for the detectionof identical sequences or a degenerate pool of possible sequences foridentification of closely related genomic sequences.

Other means for producing specific hybridization probes for nucleicacids include the cloning of nucleic acid sequences into vectors for theproduction of mRNA probes. Such vectors are known in the art and arecommercially available and may be used to synthesize RNA probes in vitroby means of the addition of the appropriate RNA polymerase as T7 or SP6RNA polymerase and the appropriate radioactively labeled nucleotides.The nucleotide sequences may be used to construct hybridization probesfor mapping their respective genomic sequences. The nucleotide sequenceprovided herein may be mapped to a chromosome or specific regions of achromosome using well known genetic and/or chromosomal mappingtechniques. These techniques include in situ hybridization, linkageanalysis against known chromosomal markers, hybridization screening withlibraries or flow-sorted chromosomal preparations specific to knownchromosomes, and the like. The technique of fluorescent in situhybridization of chromosome spreads has been described, among otherplaces, in Verma et al (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York N.Y.

Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265: 1981f). Correlation between the locationof a nucleic acid on a physical chromosomal map and a specific disease(or predisposition to a specific disease) may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier or affected individuals. Thenucleotide sequence may be used to produce purified polypeptides usingwell known methods of recombinant DNA technology. Among the manypublications that teach methods for the expression of genes after theyhave been isolated is Goeddel (1990) Gene Expression Technology, Methodsand Enzymology, Vol 185, Academic Press, San Diego. Polypeptides may beexpressed in a variety of host cells, either prokaryotic or eukaryotic.Host cells may be from the same species from which a particularpolypeptide nucleotide sequence was isolated or from a differentspecies. Advantages of producing polypeptides by recombinant DNAtechnology include obtaining adequate amounts of the protein forpurification and the availability of simplified purification procedures.

5.20 Preparation of Sequencing Chips and Arrays

A basic example is using 6-mers attached to 50 micron surfaces to give achip with dimensions of 3×3 mm which can be combined to give an array of20×20 cm. Another example is using 9-mer oligonucleotides attached to10×10 microns surface to create a 9-mer chip, with dimensions of 5×5 mm.4000 units of such chips may be used to create a 30×30 cm array. In anarray in which 4,000 to 16,000 oligochips are arranged into a squarearray. A plate, or collection of tubes, as also depicted, may bepackaged with the array as part of the sequencing kit.

The arrays may be separated physically from each other or by hydrophobicsurfaces. One possible way to utilize the hydrophobic strip separationis to use technology such as the Iso-Grid Microbiology System producedby QA Laboratories, Toronto, Canada.

Hydrophobic grid membrane filters (HGMF) have been in use in analyticalfood microbiology for about a decade where they exhibit uniqueattractions of extended numerical range and automated counting ofcolonies. One commercially-available grid is ISO-GRID™ from QALaboratories Ltd. (Toronto, Canada) which consists of a square (60×60cm) of polysulfone polymer (Gelman Tuffryn HT450, 0.45 u pore size) onwhich is printed a black hydrophobic ink grid consisting of 1600 (40×40)square cells. HGMF have previously been inoculated with bacterialsuspensions by vacuum filtration and incubated on the differential orselective media of choice.

Because the microbial growth is confined to grid cells of known positionand size on the membrane, the HGMF functions more like an MPN apparatusthan a conventional plate or membrane filter. Peterkin et al. (1987)reported that these HGMFs can be used to propagate and store genomiclibraries when used with a HGMF replicator. One such instrumentreplicates growth from each of the 1600 cells of the ISO-GRID andenables many copies of the master HGMF to be made (Peterkin et al.,1987).

Sharpe et al. (1989) also used ISO-GRID HGMF form QA Laboratories and anautomated HGMF counter (MI-100 Interpreter) and RP-100 Replicator. Theyreported a technique for maintaining and screening many microbialcultures.

Peterkin and colleagues later described a method for screening DNAprobes using the hydrophobic grid-membrane filter (Peterkin et al.,1989). These authors reported methods for effective colony hybridizationdirectly on HGMFs. Previously, poor results had been obtained due to thelow DNA binding capacity of the epoxysulfone polymer on which the HGMFsare printed. However, Peterkin et al. (1989) reported that the bindingof DNA to the surface of the membrane was improved by treating thereplicated and incubated HGMF with polyethyleneimine, a polycation,prior to contact with DNA. Although this early work uses cellular DNAattachment, and has a different objective to the present invention, themethodology described may be readily adapted for Format 3 SBH.

In order to identify useful sequences rapidly, Peterkin et al. (1989)used radiolabeled plasmid DNA from various clones and tested itsspecificity against the DNA on the prepared HGMFs. In this way, DNA fromrecombinant plasmids was rapidly screened by colony hybridizationagainst 100 organisms on HGMF replicates which can be easily andreproducibly prepared.

Manipulation with small (2-3 mm) chips, and parallel execution ofthousands of the reactions. The solution of the invention is to keep thechips and the probes in the corresponding arrays. In one example, chipscontaining 250,000 9-mers are synthesized on a silicon wafer in the formof 8×8 mM plates (15 M/oligonucleotide, Pease et al., 1994) arrayed in8×12 format (96 chips) with a 1 mM groove in between. Probes are addedeither by multichannel pipette or pin array, one probe on one chip. Toscore all 4000 6-mers, 42 chip arrays have to be used, either usingdifferent ones, or by reusing one set of chip arrays several times.

In the above case, using the earlier nomenclature of the application,F=9; P=6; and F+P=15. Chips may have probes of formula BxNn, where x isa number of specified bases B; and n is a number of non-specified bases,so that x=4 to 10 and n=1 to 4. To achieve more efficient hybridization,and to avoid potential influence of any support oligonucleotides, thespecified bases can be surrounded by unspecified bases, thus representedby a formula such as (N)nBx(N)m.

5.21 Preparation of Support Bound Oligonucleotides

Oligonucleotides, i.e., small nucleic acid segments, may be readilyprepared by, for example, directly synthesizing the oligonucleotide bychemical means, as is commonly practiced using an automatedoligonucleotide synthesizer.

Support bound oligonucleotides may be prepared by any of the methodsknown to those of skill in the art using any suitable support such asglass, polystyrene or Teflon. One strategy is to precisely spotoligonucleotides synthesized by standard synthesizers. Immobilizationcan be achieved using passive adsorption (Inouye & Hondo, 1990); usingUV light (Nagata et al., 1985; Dahlen et al., 1987; Morriey & Collins,1989) or by covalent binding of base modified DNA (Keller et al., 1988;1989); all references being specifically incorporated herein.

Another strategy that may be employed is the use of the strongbiotin-streptavidin interaction as a linker. For example, Broude et al.(1994) describe the use of Biotinylated probes, although these areduplex probes, that are immobilized on streptavidin-coated magneticbeads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Ofcourse, this same linking chemistry is applicable to coating any surfacewith streptavidin. Biotinylated probes may be purchased from varioussources, such as, e.g., Operon Technologies (Alameda, Calif.).

Nunc Laboratories (Naperville, Ill.) is also selling suitable materialthat could be used. Nunc Laboratories have developed a method by whichDNA can be covalently bound to the microwell surface termed Covalink NH.CovaLink NH is a polystyrene surface grafted with secondary amino groups(>NH) that serve as bridge-heads for further covalent coupling. CovaLinkModules may be purchased from Nunc Laboratories. DNA molecules may bebound to CovaLink exclusively at the 5′-end by a phosphoramidate bond,allowing immobilization of more than 1 pmol of DNA (Rasmussen et al.,1991).

The use of CovaLink NH strips for covalent binding of DNA molecules atthe 5′-end has been described (Rasmussen et al., 1991). In thistechnology, a phosphoramidate bond is employed (Chu et al., 1983). Thisis beneficial as immobilization using only a single covalent bond ispreferred. The phosphoramidate bond joins the DNA to the CovaLink NHsecondary amino groups that are positioned at the end of spacer armscovalently grafted onto the polystyrene surface through a 2 nm longspacer arm. To link an oligonucleotide to CovaLink NH via anphosphoramidate bond, the oligonucleotide terminus must have a 5′-endphosphate group. It is, perhaps, even possible for biotin to becovalently bound to CovaLink and then streptavidin used to bind theprobes.

More specifically, the linkage method includes dissolving DNA in water(7.5 ng/ul) and denaturing for 10 min. at 95° C. and cooling on ice for10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm₇), is thenadded to a final concentration of 10 mM 1-MeIm₇. A ss DNA solution isthen dispensed into CovaLink NH strips (75 ul/well) standing on ice.

Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC),dissolved in 10 mM 1-MeIm₇, is made fresh and 25 ul added per well. Thestrips are incubated for 5 hours at 50° C. After incubation the stripsare washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3times, then they are soaked with washing solution for 5 min., andfinally they are washed 3 times (where in the washing solution is 0.4 NNaOH, 0.25% SDS heated to 50° C.).

It is contemplated that a further suitable method for use with thepresent invention is that described in PCT Patent Application WO90/03382 (Southern & Maskos), incorporated herein by reference. Thismethod of preparing an oligonucleotide bound to a support involvesattaching a nucleoside 3′-reagent through the phosphate group by acovalent phosphodiester link to aliphatic hydroxyl groups carried by thesupport. The oligonucleotide is then synthesized on the supportednucleoside and protecting groups removed from the syntheticoligonucleotide chain under standard conditions that do not cleave theoligonucleotide from the support. Suitable reagents include nucleosidephosphoramidite and nucleoside hydrogen phosphorate.

An on-chip strategy for the preparation of DNA probe for the preparationof DNA probe arrays may be employed. For example, addressablelaser-activated photodeprotection may be employed in the chemicalsynthesis of oligonucleotides directly on a glass surface, as describedby Fodor et al. (1991), incorporated herein by reference. Probes mayalso be immobilized on nylon supports as described by Van Ness et al.(1991); or linked to Teflon using the method of Duncan & Cavalier(1988); all references being specifically incorporated herein.

To link an oligonucleotide to a nylon support, as described by Van Nesset al. (1991), requires activation of the nylon surface via alkylationand selective activation of the 5′-amine of oligonucleotides withcyanuric chloride.

One particular way to prepare support bound oligonucleotides is toutilize the light-generated synthesis described by Pease et al., (1994,incorporated herein by reference). These authors used currentphotolithographic techniques to generate arrays of immobilizedoligonucleotide probes (DNA chips). These methods, in which light isused to direct the synthesis of oligonucleotide probes in high-density,miniaturized arrays, utilize photolabile 5′-protectedN-acyl-deoxynucleoside phosphoramidites, surface linker chemistry andversatile combinatorial synthesis strategies. A matrix of 256 spatiallydefined oligonucleotide probes may be generated in this manner and thenused in the advantageous Format 3 sequencing, as described herein.

5.22 Preparation of Nucleic Acid Fragments

The nucleic acids to be sequenced may be obtained from any appropriatesource, such as cDNAs, genomic DNA, chromosomal DNA, microdissectedchromosome bands, cosmid or YAC inserts, and RNA, including mRNA withoutany amplification steps. For example, Sambrook et al. (1989) describesthree protocols for the isolation of high molecular weight DNA frommammalian cells (P. 9.14-9.23).

DNA fragments may be prepared as clones in M13, plasmid or lambdavectors and/or prepared directly from genomic DNA or cDNA by PCR orother amplification methods. Samples may be prepared or dispensed inmultiwell plates. About 100-1000 ng of DNA samples may be prepared in2-500 ml of final volume.

The nucleic acids would then be fragmented by any of the methods knownto those of skill in the art including, for example, using restrictionenzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing byultrasound and NaOH treatment.

Low pressure shearing is also appropriate, as described by Schriefer etal. (1990, incorporated herein by reference). In this method, DNAsamples are passed through a small French pressure cell at a variety oflow to intermediate pressures. A lever device allows controlledapplication of low to intermediate pressures to the cell. The results ofthese studies indicate that low-pressure shearing is a usefulalternative to sonic and enzymatic DNA fragmentation methods.

One particularly suitable way for fragmenting DNA is contemplated to bethat using the two base recognition endonuclease, CviJI, described byFitzgerald et al. (1992). These authors described an approach for therapid fragmentation and fractionation of DNA into particular sizes thatthey contemplated to be suitable for shotgun cloning and sequencing. Thepresent inventor envisions that this will also be particularly usefulfor generating random, but relatively small, fragments of DNA for use inthe present sequencing technology.

The restriction endonuclease CviJI normally cleaves the recognitionsequence PuGCPY between the G and C to leave blunt ends. A typicalreaction conditions, which alter the specificity of this enzyme(CviJI**), yield a quasi-random distribution of DNA fragments form thesmall molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992)quantitatively evaluated the randomness of this fragmentation strategy,using a CviJI** digest of pUC19 that was size fractionated by a rapidgel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed thatCviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, andthat new sequence data is accumulated at a rate consistent with randomfragmentation.

As reported in the literature, advantages of this approach compared tosonication and agarose gel fractionation include: smaller amounts of DNAare required (0.2-0.5 ug instead of 2-5 ug); and fewer steps areinvolved (no preligation, end repair, chemical extraction, or agarosegel electrophoresis and elution are needed). These advantages are alsoproposed to be of use when preparing DNA for sequencing by Format 3.

Irrespective of the manner in which the nucleic acid fragments areobtained or prepared, it is important to denature the DNA to give singlestranded pieces available for hybridization. This is achieved byincubating the DNA solution for 2-5 minutes at 80-90° C. The solution isthen cooled quickly to 2° C. to prevent renaturation of the DNAfragments before they are contacted with the chip. Phosphate groups mustalso be removed from genomic DNA by methods known in the art.

5.23 Preparation of DNA Arrays

Arrays may be prepared by spotting DNA samples on a support such as anylon membrane. Spotting may be performed by using arrays of metal pins(the positions of which correspond to an array of wells in a microtiterplate) to repeated by transfer of about 20 nl of a DNA solution to anylon membrane. By offset printing, a density of dots higher than thedensity of the wells is achieved. One to 25 dots may be accommodated in1 mm², depending on the type of label used. By avoiding spotting in somepreselected number of rows and columns, separate subsets (subarrays) maybe formed. Samples in one subarray may be the same genomic segment ofDNA (or the same gene) from different individuals, or may be different,overlapped genomic clones. Each of the subarrays may represent replicaspotting of the same samples. In one example, a selected gene segmentmay be amplified from 64 patients. For each patient, the amplified genesegment may be in one 96-well plate (all 96 wells containing the samesample). A plate for each of the 64 patients is prepared. By using a96-pin device, all samples may be spotted on one 8×12 cm membrane.Subarrays may contain 64 samples, one from each patient. Where the 96subarrays are identical, the dot span may be 1 mm² and there may be a 1mm space between subarrays.

Another approach is to use membranes or plates (available from NUNC,Naperville, Ill.) which may be partitioned by physical spacers e.g. aplastic grid molded over the membrane, the grid being similar to thesort of membrane applied to the bottom of multiwell plates, orhydrophobic strips. A fixed physical spacer is not preferred for imagingby exposure to flat phosphor-storage screens or x-ray films.

The present invention is illustrated in the following examples. Uponconsideration of the present disclosure, one of skill in the art willappreciate that many other embodiments and variations may be made in thescope of the present invention. Accordingly, it is intended that thebroader aspects of the present invention not be limited to thedisclosure of the following examples. The present invention is not to belimited in scope by the exemplified embodiments which are intended asillustrations of single aspects of the invention, and compositions andmethods which are functionally equivalent are within the scope of theinvention. Indeed, numerous modifications and variations in the practiceof the invention are expected to occur to those skilled in the art uponconsideration of the present preferred embodiments. Consequently, theonly limitations which should be placed upon the scope of the inventionare those which appear in the appended claims.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

6.0 EXAMPLES Example 1 Isolation of SEQ ID NO: 1-7 from a cDNA Librariesof Human Cells

A plurality of novel nucleic acids were obtained from a cDNA libraryprepared from human testis cells (Hyseq clone identification numbers2880984 and 2881695), from human fetal skin (Hyseq clone identificationnumber 15375176), adult spleen (Hyseq clone identification number14856094), and human endothelial cells (Hyseq clone identificationnumbers 13804756, 13687487, 13804756) using standard PCR, sequencing byhybridization sequence signature analysis, and Sanger sequencingtechniques. The inserts of the library were amplified with PCR usingprimers specific for vector sequences flanking the inserts. Thesesamples were spotted onto nylon membranes and interrogated witholigonucleotide probes to give sequence signatures. The clones wereclustered into groups of similar or identical sequences, and singlerepresentative clones were selected from each group for gel sequencing.The 5′ sequence of the amplified inserts was then deduced using thereverse M13 sequencing primer in a typical Sanger sequencing protocol.PCR products were purified and subjected to fluorescent dye terminatorcycle sequencing. Single-pass gel sequencing was done using a 377Applied Biosystems (ABI) sequencer. These inserts was identified as anovel sequence not previously obtained from this library and notpreviously reported in public databases. These sequences are designatedas SEQ ID NO: 1-7 in the attached sequence listing.

Example 2 Assemblage of SEQ ID NO: 8 and 9

The novel nucleic acids (SEQ ID NO: 8 and 9) of the invention wereassembled from sequences that were obtained from a cDNA library bymethods described in Example 1 above, and in some cases sequencesobtained from one or more public databases. The sequence was assembledusing an EST sequence (SEQ ID NO: 2) as a seed. Then a recursivealgorithm was used to extend the seed EST into an extended assemblage,by pulling additional sequences from different databases (i.e., Hyseq'sdatabase containing EST sequences, dbEST version 114, gb pri 114, andUniGene version 101) that belong to this assemblage. The algorithmterminated when there was no additional sequences from the abovedatabases that would extend the assemblage. Inclusion of componentsequences into the assemblage was based on a BLASTN hit to the extendingassemblage with BLAST score greater than 300 and percent identitygreater than 95%. SEQ ID NO: 8 was further manually edited to obtain SEQID NO: 9. FIG. 1 shows the alignment of SEQ ID NO. 9 with SEQ ID NO.1-7.

The nearest neighbor result for the assembled sequence (SEQ ID NO. 8)was obtained by a FASTA version 3 search against Genpept release 114,using Fastxy algorithm. Fastxy is an improved version of FASTA alignmentwhich allows in-codon frame shifts. The nearest neighbor result showedthe closest homologue for each assemblage from Genpept (and contains thetranslated amino acid sequences for which the assemblage encodes). Thenearest neighbor results is set forth below: Accession Smith- % No.Description Waterman Score Identity AB016768 Mus musculus thrombospondin56 43.750 type 1 domain

The predicted amino acid sequence for SEQ ID NO: 8 was obtained by usinga software program called FASTY (available fromhttp://fasta:bioch.virginia.edu) which selects a polypeptide based on acomparison of translated novel polynucleotide to known polynucleotides(W.R. Pearson, Methods in Enzymology, 183:63-98 (1990), incorporatedherein by reference).

For SEQ ID NO: 8: Amino acid segment con- taining signal peptide (A = Alanine, C = Cysteine, D =  Aspartic Acid, E = Glutamic Acid, F= Phenylalanine, G = Glycine, H = Histidine, I = Isoleucine, K = Lysine, L = Leucine, M =  Predicted Methionine, N = Asparagine,beginning Predicted end P = Proline, Q =  nucleotide nucleotideGlutamine, R = Arginine, location location S = Serine, T = Threonine,corresponding corresponding V = Valine, W = Tryptophan, to first tofirst Y = Tyrosine, X =  amino acid amino acid Unknown, * = Stop Codon,residue of residue of / = possible nucleotide amino acid amino aciddeletion, \ = possible sequence sequence nucleotide insertion) 575 1054CTKCKADCDTCFNKNFCTKCKSGFYLH LGKCLDNCPEGLEANNHTMECVSIVHCEVSEWNPWSPCTKKGKTCGFKRGTETR VREIIQHPSAKGNLCPPTNETRKCTVQRKKCQKGERGKKGRERKRKKPNKGESK EAIPDSKSLESSKEIPEQRENKQQQ (SEQ ID NO: 14)

Example 3 Assemblage of SEQ ID NO: 10

A polypeptide (SEQ ID NO: 10) was predicted to be encoded by SEQ ID NO:9 as set forth below. The polypeptide was predicted using a softwareprogram called BLASTX which selects a polypeptide based on a comparisonof translated novel polynucleotide to known polynucleotides. The initialmethionine starts at position 291 of SEQ ID NO: 9 and the putative stopcodon, TAG, begins at position 1107 of the nucleotide sequence.

Example 4 Cloning of Stem Cell Growth Factor-Like Gene; and Expressionand Purification of Stem Cell Growth Factor-Like Protein

Stem cell growth factor-like polynucleotide (SEQ ID NO: 11 or 12) wascloned by PCR into pIB/V5-His TOPO TA cloning vector (Invitrogen) fromHyseq 's full-length stem cell growth factor-like clone. Stem cellgrowth factor-like gene was further subcloned into pCDNA3.1-Myc-Hisvectors (Invitrogen) and expressed with or without V5-His tag. Insectcells (high Five, Invitrogen) were transfected with stem cell growthfactor-like gene with the His-5 tag by using the InsectSelect system(Invitrogen) using manufacturer's suggested protocols. Stem cell growthfactor-like protein was purified from the cell media by a combination ofpH adjustment, cation exchange chromatography, and affinitychromatography as described below. Briefly, the pH of the medium wasadjusted to 7.0 and the protease inhibitors PMSF and EDTA were added.Column chromatography purification was performed on Pharmacia Aktainstrument system at room temperature using sequential removal ofcontaminants on appropriately sized columns of SP-Sepharose Fast Flow,Hitrap heparin Sepharose, and Ni-NTA resins. Column elution fractionswere analyzed by separation on 16% SDS-PAGE gels, transfer to Immobilinmembranes (Millipore), and detection of the tagged protein by anti-V5antibody using manufacturer's protocols. Fractions containing stem cellgrowth factor-like activity eluted from Ni-NTA column were pooled andequilibrated with PBS and stored at −80° C. until analyzed for stem cellgrowth factor-like activity.

Example 5 Expression of Stem Cell Growth Factor-Like Protein in PrimaryHuman Cells

The product of the secondary nested PCR from Marathon spleen library(SEQ ID NO: 11 or 12) or any other polynucleotide encoding stem cellgrowth factor-like polypeptide were cloned into MSCV retroviral vector(Clontech) into suitable cloning sites using appropriate forward andreverse PCR primers. This retroviral vector was then transfected usingFUGENE-6 transfection reagent into packaging cell lines to producesuitably large quantities of retrovirus that will have the stem cellgrowth factor-like DNA cloned in it. Retrovirus containing supernatantswere prepared from packaged cell lines and mixed with stromal or stemcells. Upon retrovirus transduction these transduced cells may expressthe stem cell growth factor-like protein.

Example 6 Assay for Growth and Differentiation of Stem Cells UsingCoculture Assay

1×10⁴ mouse stem cells were co-cultured with 1×10⁴ stem cell growthfactor-like polynucleotide-transduced stromal cells or vector-transducedstromal cells (produced by Example 5) in the serum-free medium. On dayseven, IL-3 (10 ng/ml) and IL-6 (10 ng/ml) were added as additionalgrowth factors. Cultures were monitored microscopically every day. Afterappropriate further incubation, cells were harvested, and counted usinghemacytometer. Results from one experiment are presented in the tablebelow: Day 15 Day 18 (approximate (approximate number of number ofConditions cell/ml) cell/ml) Vector-transduced stroma  30000 105000cells + stem cells Stem cell growth factor 405000 825000polynucleotide-trans- duced stroma cells + stem cells

Example 7 Assay for Proliferation and Differentiation of Stem Cells

CD34⁺ hematopoietic stem cells (HSC) were purified from mobilizedperipheral blood (purchased from ALLCells). CD34⁺ cells were purified bypositively selecting cells using Miltenyi breads (Miltenyi). Stem cellswere plated in 96-well plates at 10³/well. Purified stem cell growthfactor-like protein and other hematopoietic cytokines (purchased from R& D systems), and the combinations thereof were added to the culturesfor assessing the stem cell growth factor activity. The growth anddifferentiation of stem cells were examined 5 days after culture bylight microscope. The results of six experiments are shown in the tablebelow, wherein positive effect of stem cell growth factor protein wasobserved in three out of six experiments.

Abbreviations: Stem cell growth factor-like protein ═SCGF;Interleukin-3=IL-3; thrombopoietin=TPO; Fms-like tyrosine kinase-3ligand=flt-3 ligand (+) indicates growth and/or differentiation of stemcells; (−) indicates no growth or differentiation and loss of viabilityof stem cells. Growth and Growth and morphological morphological changeschanges Growth factor(s) added Experiment 1 Experiment 2 None (−) (−)Stem cell growth factor (50 ng/ml) (−) (−) IL-3 (10 ng/ml) (+/−) (+)SCGF (50 ng/ml) + IL-3 (10 ng/ml) (+) (+) Growth and Growth and Growthand morphological morphological morphological changes changes changesGrowth factor(s) added Experiment 3 Experiment 4 Experiment 5 None (−)(−) (−) Stem cell growth (−) (−) (−) factor (50 ng/ml) TPO (100 ng/ml)(−) (−) (−) kit ligand (50 ng/ml) + (−) (−) (−) flt-3 ligand (50 ng/ml)kit ligand (50 ng/ml) + (+) (−) (−) flt-3 ligand (50 ng/ml) + SCGF (50ng/ml) Growth and morphological changes Growth factor(s) addedExperiment 6 None (−) Stem cell growth factor (50 ng/ml) (−) kit ligand(50 ng/ml) (−) flt-3 ligand (50 ng/ml) (−) kit ligand (50 ng/ml) + flt-3ligand (50 ng/ml) (−) kit ligand (50 ng/ml) + flt-3 ligand (50 ng/ml) +SCGF (+) (50 ng/ml)

Example 8 Establishment of Stromal Cell Strain Derived from Mouse AGM

(1) Isolation of AGM Region from Fetal Mouse

C3H/HeNSLc mouse of both genders (purchased from Japan SLC INC.) wasbred under a SPF (specific pathogen-free) circumstance. One or twofemale mice and one male mouse were reared in the same cage over anight. In the next morning, the female mice in which the existence of avaginal plug was confirmed were transferred to other cages and breeded.The day when the existence of the vaginal plug was confirmed was definedto be the 0.5th day of pregnancy. On the 10.5th day of the pregnancy,after mouse was sacrificed by cervical dislocation, fetuses wereextirpated. Isolation of AGM regions was performed according to themethod by Godin et al. (Godin, I., Proc. Natl. Acad. Sci. U.S.A., 92:773-777, 1995) and the method by Medvinsky et al. (Medvinsky, A. L.,Blood, 87: 557-565, 1996). The fetuses were placed in a culture dishesto which PBS(−) (phosphate buffered saline) (produced by Nissui Seiyaku)was added in a volume just sufficient to cover them. After the AGMregions were carefully excised so as not to include other regions undera stereoscopic microscope, they were put in another 24-well culture dish(Nunc).

(2) Establishment of Cell Lines Derived from AGM

One drop of MEM medium (Sigma) containing 10% FCS (Hyclone) was added tothe AGM regions in the 24-well culture dish (Nunc), and AGM regions werecultured in incubator overnight. The cultures were performed in the MEMmedium (Sigma) including 10% FCS (Hyclone) at 37° C., in an atmosphereof 5% CO₂, and at a humidity of 100%. When the cells corresponding tothe AGM regions adhered to the culture dish due to overnightcultivation, two milliliters of MEM medium containing 10% FCS wasfurther added. Stromal cells began to appear around the AGM regiontissue fragment after the continuous cultivation. After one-weekcultivation, adhesive cells were trypsinized (0.05% trypsin in PBScontaining 0.53 mM EDTA (Gibco BRL) at 37° C. for three to five minutes)and dispersed. The stromal cells were then washed twice with the medium,and seeded on 6-well culture dish (Nunc). On the next day, the cellswhich did not adhered to the culture dish and the medium were removed,and then, fresh medium were added. Two weeks after transfer, the cellsin the 6-well culture dish were γ-ray irradiated at 900 Rad to eliminateendogenous hematopoietic cells. Although attempts of the direct cellcloning by limiting dilution from this culture system was failed, sothat no cell proliferation was observed. Then, attempts were madeaccording to as follows: after adaptation of cells so as to be able toproliferate from a small number of cells by increasing the number ofseeded cells in one well, the cells were cloned by limiting dilution.

That is, the AGM was extirpated and cultured in the same manner asdescribed above. The culture system two weeks after the γ-ray radiationwas trypsinized (0.05% trypsin in PBS containing 0.53 mM EDTA at 37° C.for three to five minutes) and the cells were suspended, so that thecells were seeded in a 24-well culture dish ranging from 50 to 100cells/well. After the culture was continued for three weeks, the cellswere seeded in a 96-well culture dish (Nunc) by means of limitingdilution so as to be 0.3 cells/well. The cells which were derived fromthe well seeded only one cell and proliferated were allowed to enlargeculture. As a result, the cells were successfully cloned to obtainfibroblast like cells and cobble stone like cells.

CD34 positive cell fraction derived from the human cord blood wasco-cultured with the fibroblast like cells for two weeks. Colony formingcells could not be found in the co-culture system with the fibroblastlike cells. Then, the similar examination was performed for seven cellclones showing cobble stone like morphotype. Three clones havingactivity to proliferate and support the human hematopoietic stem cellswere obtained and were named AGM-s1, AGM-s2, and AGM-s3.

Example 9 Preparation of Hematopoietic Stem Cells from Mouse Bone Marrow

The bone marrow was collected from the femur of C57BL/6-Ly5.1 pep (weekages ranging from eight to ten, and male) (the gift from Professor K.Nakauchi, University of Tsukuba), and suspended in PBS. After the mousebone marrow mononuclear cells were concentrated by specific gravitycentrifugation according to the usual method (S. Kouzu, Fundamentaltechniques for immunology, YODOSHA, 1995), the cells were suspended withstaining buffer (PBS containing 5% FCS and 0.05% NaN₃).

The most immature hematopoietic stem cell fraction was obtained asfollows (Osawa, M. et al., Science 273: 242-245, 1996).

The mononuclear cells were incubated with biotylated anti-lineagemonoclonal antibodies (CD45R, CD4, CD8, Gr-1, Ter119, and CD11c,purchased from Pharmingen), fluorescein isothiocyanate (FITC)-anti-CD34,phycoerythrin (PE)-anti-Sca-1, and allophycocyanin (APC)-anti-c-Kit for20 min on ice. After the stained cells were washed twice with stainingbuffer, CD34 negative, Sca-1 positive, c-Kit positive, and Lin negativecells were isolated on a FACS Vantage (Becton Dickinson).

Example 10 Subcloning of Mouse Stromal Cell Strain and Assessment of anActivity to Support the Hematopoietic Stem Cells of a Variety of CellStrains

(1) Subcloning of Mouse Stromal Cell Strain

1) Isolation of AGM-s3 Subclone

Stromal cell strain AGM-s3 derived form AGM which was subcultured inMEMα medium (GIBCO BRL) including non-active 10% FCS (bovine fetalserum, Hyclone) was suspended in PBS containing 5% FCS (PBS-FCS). Clonesorting was performed in a 96-well culture dish (Falcon) at onecell/well using a cell sorter (FACS Vantage; Becton Dickinson). Amongcells in the 96 wells, cultures of the cells which proliferated wereexpanded, so that thirteen kinds of AGM-s3 subclones were obtained. Theactivity to support the hematopoietic cells of these AGM-s3 subcloneswere assessed.

2) Isolation of Human Cord Blood CD34 Positive Stem Cell

The human cord blood was collected at normal delivery according to thecriteria approved by Drug Discovery Institute, Ethics committee, KirinBrewery Co., LTD. The cord blood was collected using a syringe addedwith heparin so as not to coagulate. The heparin treated cord blood wasoverlaid on Lymphoprep (NYCOMED PHARMA), and mononuclear cells wereseparated by centrifugation (at 400G, at room temperature, and for 30minutes). Erythrocytes contaminated in the mononuclear cell fractionwere lyzed by treatment with ammonium chloride buffer solution (0.83%NH₄Cl-Tris HCl, 20 mM, pH 6.8) at room temperature for two minutes.After the mononuclear cells were washed with PBS-FCS, ten milligrams ofhuman IgG was added and allowed to stand on ice for ten minutes. Then,the cells were further washed with PBS-FCS, added with biotinylatedantibodies against the antigens specific to the human differentiatedblood cells that is, CD2, CD11c (purified from ATCC hybridoma), CD19(Pharmingen), CD15, and CD41 (Leinco Technologies Inc.), and theantibody against Glycophorin A (Cosmo Bio), and allowed to stand on icefor 20 min. After washing with PBS-FCS, the cells were suspended in onemilliliter of PBS containing 5% FCS, 10 mM EDTA, and 0.05% NaN₃(PBS-FCS-EDTA-NaN₃), added with magnetic beads bound with streptavidin(BioMag. Per Septive Diagnostics), and allowed to stand on ice for 40min. The differentiated blood cells which expressed differentiationantigens were removed using a magnetic separator (Dynal MPC-1 Dynal).FITC labeled CD34 antibody (Immunotech S. A., Marseilles, France) wereadded to the remaining differentiated blood cell antigen negative cellfraction. After incubation on ice for 20 min., CD34 positive fractionwas recovered using a cell sorter. This cell fraction was defined as ahematopoietic stem cell fraction derived from the human cord blood.

3) Co-culture of the Human Hematopoietic Stem Cells and AGM-s3 Subclone

After 13 kinds of AGM-s3 subclones or stromal cell strain MS-5 derivedfrom the mouse bone marrow were seeded in a 24-well culture dish(Falcon) at 1×10⁴ cells/well, and cells were cultured in one milliliterof MEMα medium containing 10% FCS until the cells covered all over thebottom surfaces of the wells. CD34 positive hematopoietic stem cellsderived from the human cord blood were sorted on the above describedstromal cells at 500 cells/well, and co-cultured in one milliliter ofMEMα medium containing 10% FCS. One week after the initiation of theco-culture, one milliliter of the same medium was further added. Twoweeks after the initiation of the co-culture, the stromal cells and thehuman blood cells were trypsinized (0.05% trypsin in PBS containing 0.5mM EDTA (GIBCO BRL) at 37° C. and standing for two to five min.) anddispersed from the culture dish. Activities to support the hematopoieticstem cells were assessed with a colony assay.

4) Assessment of Proliferation Statuses of the Hematopoietic Stem Cellsand Hemopoietic Precursor Cells by Clonogenic Assay

The cells which proliferated in the above described co-culture systemwere appropriately diluted, added to one milliliter of methylcelluloseculture system, and analyzed in triplicate. The analysis using themethylcellulose culture system were performed using a 6-well culturedish (Falcon) in the presence of 10 ng/ml of human SCF, human IL-3,human IL-6, human G-CSF, human TPO, and EPO at 2 IU/ml to MethoCultH4230 (Stem Cell Technologies Inc.). All of a variety of the abovedescribed hematopoietic factors were recombinants and pure. Two weeksafter the culture, developed colonies were observed under a microscopeand counted numbers of CFU-GM (granulocyte-macrophage differentiatingseries), BFU-E (erythroid burst forming unit), and CFU-E mix(erythrocyte mixed differentiating series).

FIG. 4 shows the results from two-week co-culture of the CD34 positivehematopoietic stem cells and AGM-s3 subclones A9, A7, or D11. As aresult of the co-culture, A9 and D11 subclones among 13 kinds of AGM-s3subclones supported proliferation of all three lineages of CFU-GM,BFU-E, and CFU-E mix. Especially, although BFU-E and CFU-E mix, that is,the precursor cells of an erythrocytes were hardly to be supported inusual, they proliferated in the co-culture system with A9 or D11 cells.The results showed that proliferation or maintenance of thehematopoietic stem cells or the hemopoietic precursor cells occurred inthe co-culture with A9 or D11 cells and the precursor cells of theerythrocyte were continuously supplied. In contrast, although cellularmorphology of A7 was similar to that of A9, A7 did not support CFU-GM,BFU-E, and CFU-E mix.

5) Comparison of an Activity to Support the Human Hematopoietic StemCells Between A9 and a Stromal Cell Strain OP9 Derived from Mouse Fetus

Comparison of activities to support the CD34 positive hematopoietic stemcells derived from the human cord blood between AGM-s3 subclones A9 andA7, and a stromal cell line OP9 derived from mouse fetus were performedwith CFU-GM, BFU-E, CFU-E and CFU-E mix as indexes using the abovedescribed method. FIG. 5 shows the results from the two-week co-culture.In the A7 cell culture system, CFU-GM, BFU-E, and CFU-E weresignificantly decreased and CFU-E mix was completely disappeared. Incontrast, with OP9 cells, a variety of blood cell precursor cellsincluding CFU-E mix were supported, although the supporting ability wasless than that of A9 cells. Therefore, OP9 cells were clear to possessthe activity to support the hematopoietic stem cells.

(2) Assessment of Activities to Support the Hematopoietic Stem Cells ina Variety of Cell Strains

The above described stromal cell lines (AGM-s3-A9, AGM-s3-A7, andAGM-s3-G1), 3T3Swiss (ATCC), OP9 (RCB1124, RIKEN Cell Development Bank),and NIH3T3 (ATCC) were seeded in a 24-well culture dish (Falcon) at5×10⁴ cells/well. The cell lines were cultured in MEMα medium (GIBCOBRL) containing non-active 10% FCS (bovine fetal serum, Hyclone) for oneday and allowed to proliferated until the cells covered all over thebottom surfaces of the wells. Then, the medium was replaced to onemilliliter of fresh medium, thirty cells of the mouse hematopoietic stemcells (derived from C57BL/6-Ly5.1) obtained in Example 9 were sorted onthis cell layer, and co-culture was initiated.

On seventh day of the cultivation, the cells were trypsinized (0.05%trypsin in PBS containing 0.5 mM EDTA (GIBCO BRL) at 37° C. for two tofive minutes) and dispersed and all the cells on the culture dish wererecovered. The recovered whole cells of each cell line and whole bonemarrow cells at 200,000 cells (derived from C57BL/6-Ly5.2 mouse, CharlesRiver) were transplanted into the C57BL/6-Ly5.2 mice (eight weeks ageand male, Charles River) irradiated with X-ray at 8.5 Gy through thetail vein. After the transplantation, the peripheral blood was collectedfrom the retro-orbital sinus at intervals, and calculated the ratio of acell number derived from the C57BL/6-Ly5.1 prep mouse with FACS. Theperipheral blood was analyzed according to the usual method (S. Kouzu,Fundamental techniques for immunology, YODOSHA, 1995). Three hundredsand fifty μL of distilled water was added to 50 μL of the peripheralblood, allowed to stand for 30 sec. so as to lyze the erythrocytes.Then, PBS at twice concentrations was added and centrifuged, so that thewhite blood cells were recovered. After the cells were washed once usingthe staining buffer (PBS containing 5% FCS and 0.05% NaN₃), anti-CD16antibody, Ly5.1 (CD45.1) antibody labeled with FITC, Gr-1 and CD11cantibodies labeled with phycoerythrin, and CD45R (B220) antibody andCD90 (Thy1) antibody labeled with allophycocyanin (all of these werepurchased from Pharmingen) were added. After these cells were allowed tostand for reaction in the ice bath for 30 min., they were washed withthe staining buffer and FACS analysis was performed.

Expansion in the number of cells capable of reconstitution during thehematopoietic stem cell culture was assessed by calculating theproportions of Ly5.1 positive cells in the Gr-1 or CD11c positive cells(myeloid cells) or Ly5.1 positive cells in the CD90 or CD45R positivecells (lymphoid cells) in the peripheral blood at intervals posttransplantation.

FIG. 6 shows the results. When the cells were co-cultured withAGM-s3-A9, OP9, and 3T3Swiss cells, high chimerism of donor cells weremaintained after the transplantation. Therefore, these stromal cellswere considered to have a high activity to support the hematopoieticstem cells. In contrast, when the cells were co-cultured with AGM-s3-A7,AGM-s3-G1, and NIH3T3 cells, high chimerism were not observed in thetransplanted cells. Therefore, these stromal cells had low activity tosupport the hematopoietic stem cells or the hemopoietic progenitorcells.

Example 11 Isolation of Mouse SCR-1 Fragment

Total RNA was prepared from AGM-s3-A9 cells at 1.4×10⁸ cells dissolvedin 20 mL of ISOGEN (Nippon gene, Japan) according to the attachment.Messenger RNA was purified from one milligram of the total RNA accordingto the protocol of the mRNA purification kit (Amersham Pharmacia,U.S.A.). cDNA was synthesized from this mRNA by oligo-dT primed withSuperScript Plasmid System (GIBCO Lifetech, U.S.A.) and inserted intopSPORT1 (GIBCO Lifetech, U.S.A.). An AGM-s3-A9 cell specific cDNA clonewas obtained from this library with SBH method (Hyseq, U.S.A.). Anucleotide sequence of the clone was determined using ABI377 DNAsequencer (Perkin Elmer, U.S.A.). The obtained sequence was analyzed byhomology search, so that the gene was identified as a novel gene SCR-1.The nucleotide sequence obtained was nucleotide numbers 1032 to 1484 ofSEQ ID NO: 31.

Example 12 Whole Cloning of Mouse SCR-1

Total RNA was prepared from AGM-s3-A9 cells at 1.4×10⁸ cells dissolvedin 20 mL of ISOGEN (Nippon gene, Japan) according to the attachment.Messenger RNA was purified from one milligram of the total RNA accordingto the protocol of the mRNA purification kit (Amersham Pharmacia,U.S.A.). cDNA library was constructed from 2 mg of prepared mRNA usingSMART cDNA library construction kit (CLONTECH, U.S.A.) according to theattachment. This library included about 400,000 kinds of independentclones in total and divided into 15 fractions. The fraction containingSCR-1 cDNA clone was identified by PCR using the following conditions.

The following primers were synthesized based on the gene fragmentsequence obtained in Example 11 PCR at 35 cycles was performed usingeach fraction of AGM-s3-A9 cDNA library as a template, one cycle being astep performed at 94° C. for 30 seconds, at 55° C. for 30 seconds, and72° C. for one minute. SCR-1F1: AGTACAAAGAAAGAAGTGTTC (SEQ ID NO: 35)SCR-1R1: TGAGTCTACAGTAACCTCGCA (SEQ ID NO: 36)

The PCR products were subjected to a 2% agarose gel electrophoresis, andthe fraction in which a PCR product had an expected size was identified.Two positive fractions were seeded on petri dishes at a diameter of 15cm at 50,000 plaque, each fractions being seeded on two petri dishes.After incubating the dishes at 37° C. for 10 hours, each plaque wastransferred to a Biodyne nylon filter (Pall, U.S.A.). DNAs on the nylonfilters were immobilized according to the attachment. Screening wasperformed using a ³²P labeled DNA probe.

The probe was prepared as follows. PCR at 35 cycles was performed usingSCR-1R1 and T7 primer (TAATACGACTCACTATAGGG) (SEQ ID NO: 37), and aplasmid including the gene fragment obtained in Example 11 as atemplate, one cycle being a step performed at 94° C. for 30 seconds, at55° C. for 30 seconds, and 72° C. for one minute. The PCR products weresubjected to a 2% agarose gel electrophoresis, and the amplifiedfragment was purified using JETSORB (GENOMED Ger.). ³²P labeled DNAprobe was prepared using Megaprime labeling kit (Amersham PharmaciaU.S.A.) and 25 ng of purified PCR fragment as a template.

Hybridization using ExpressHybSolution (CLONTECH, U.S.A.) and washingwere performed according to the attachment. X-ray films (Fuji Photo FilmCo. Ltd., Japan) were exposed to the hybridized nylon filters for oneday and developed using a Fuji film auto-developer apparatus. Based onthe analyzed results, the plaque which corresponded to the spot stronglyexposed was scratched from the petri dish. The plaque was again seededon a petri dish at a diameter of 10 cm so as to generate about 200plaques. Screening was performed again according to the method describedabove, so that a single plaque was isolated. The obtained phage clonewas introduced into E. coli BM25.8 strain according to the attachment ofthe SMART cDNA library constructing kit, so that it was excised in vivoin E. coli BM25.8 strain. The infected E. coli was cultured on LB agarmedium added with 50 mg/mL of ampicillin until colonies were formed. Asingle colony was seeded in three milliliters of LB medium containing 50mg/mL of ampicillin and cultured overnight at 30° C. About 10 mg ofplasmid was purified from the cultured cells using RPM Kit (BIO101,U.S.A.). The sequence of the both ends of the inserted fragment wasdetermined using λTrip1Ex5′LD-Insert Screening Amplimer(CTCGGGAAGCGCGCCATTGTGTTGGT: SEQ ID NO: 30 CLONTECH, U.S.A;).by ABI377 DNA sequencer. The clone was found to include cDNA which has anucleotide sequence beginning from 1 in SEQ ID NO: 31. After the wholenucleotide sequence of the inserted cDNA was determined using ABI377 DNAsequencer, the nucleotide sequence of SEQ ID NO: 31 was confirmed. Aminoacid sequences predicted from the above nucleotide sequence were shownin SEQ ID NO: 31 and SEQ ID NO: 32.

The plasmid including DNA with the nucleotide sequence of SEQ ID NO: 31has been internationally deposited in National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology (Zipcode 305-8566; Higashi 1-1-3, Tsukuba, Ibaraki, Japan) on Jun. 26, 2000,and the registered number was given to be FERM BP-7198.

Example 13 Cloning of Human SCR-1

Based on the nucleotide sequence of mouse SCR-1, the database of GenBank(NCBI, U.S.A.) was searched using Blast. A homologous nucleotidesequence with mouse SCR-1 was found (Accession Nos. AI872133 andAW316562). The following primers were synthesized using this sequencederived from human. h782F1: TCGCGGCGATGCCAGCCACCCCAG (SEQ ID NO: 38)h782F2: AGCACGCCTATCGGATGTGAGAGGAGAAGT (SEQ ID NO: 39) h782R1:CTATTAACAAATATATTTATTGTGGTGGCT (SEQ ID NO: 40) h782R2:TGGTGGCTTTCTCCCCTACTAGATATACCT (SEQ ID NO: 41)

cDNA was synthesized from 3 μg of mRNA derived from the placenta and theskeletal muscle (CLONTECH, U.S.A.) using oligo-dT primer and reversetranscriptase (SuperscriptII, GIBCO-BRL). PCR was performed using thiscDNA as a template; h782F1, h782F2, h782R1, or h782R2 as a primer; andPlatinum Pfx DNA Polymerase (GIBCO Lifetech, U.S.A.). As a result, anamplified fragment was obtained from each organ. Among them, the PCRfragment derived from the placenta was ligated to pCR-Blunt vector(Invitrogen, U.S.A.), and the gene was introduced into E. coli DH5a.Then, the transferred E. coli was seeded on LB agar medium containing100 mg/ml of ampicillin, so that colonies were formed. Each of isolated16 colonies was added to 10 ml of PCR reaction solution, and treated at94° C. for five minutes. Then, PCR at 35 cycles was performed, one cyclebeing a step performed at 94° C. for 30 seconds, at 55° C. for 30seconds, and 72° C. for one minute. T7 primer or SP6 primer(GATTTTAGGTGACACTATAG) (SEQ ID NO: 42) was used as a primer at a finalconcentration of 0.2 mM. The PCR products were subjected to a 2% agarosegel electrophoresis. After the amplified fragment was confirmed,sequences of the three confirmed fragments were determined using ABI377DNA sequencer. A nucleotide sequence of the obtained cDNA (SEQ ID NO:33) was confirmed to be a human orthologous to that of mouse SCR-1.

The plasmid including DNA with the nucleotide sequence of SEQ ID NO: 33has been internationally deposited in National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology (Zipcode 305-8566; Higashi 1-1-3, Tsukuba, Ibaraki, Japan) on Jun. 26, 2000,and the registered number was given to be FERM BP-7197.

With respect to mouse SCR-1 and human SCR-1, when the establisheddatabase was searched, a human gene sequence having unknown function wasfound (WO98/49302). The homology of coding regions of these genes isshown in Table 1. The comparison of the homology was performed using ahomology search function of DNAIS-Mac version 3.7 and calculating withthe default settings of the software (nucleic acid Mode: Normal, Range:All (1-819 base), Cutoff: 45, Ktup: 4, amino acid Range: All (1-273a.a.), Cutoff: 45, Ktup: 2). In this method, since only high homologousregions are used for calculation, low homologous regions, locating at anend, are excluded for calculation. TABLE 1 Mouse SCR-1 Gene havingunknown function Human SCR-1 88.5 (87.1) 98.5 (100)  Mouse SCR-1 87.2(85.1)The upper numbers show the homology in nucleotide level.The lower numbers (parenthesize) show the homology in amino acid level.

Human SCR-1 and the gene having unknown function had the samenucleotides from the initiation codon to the first nucleotide of the266th codon (nucleotide number 1054 in SEQ ID NO: 33) in the codingregion. However, the down stream sequences thereof were not identical.The nucleotide sequence and the amino acid sequence of this nonidenticalregion in this gene having unknown function were shown in SEQ ID NO: 45and SEQ ID NO: 46, respectively. The first nucleotide in SEQ ID NO: 45corresponded to the nucleotide number 1054 in SEQ ID NO: 33, and thesewere identical. One nucleotide was nonidentical at the positioncorresponding to the 567th nucleotide in SEQ ID NO: 33 in both genes.

Example 14 Study of the Expression Region of SCR-1

Northern blot analysis was performed using probes used in Example 13.Hybridization was performed with respect to Northern blots of Human MTNBlot I, II, III, Immune System II, Mouse MTN Blot (CLONTECH, U.S.A.).The hybridization was performed using ExpressHyb Hybridization Solution(CLONTECH, U.S.A.) according to the supplier's instruct ion. Afterprehybridization at 68° C. for two hours, a labeled probe was added.Hybridization was further performed at 68° C. for 18 hours. Washing ofthe filter was performed at room temperature in 2×SSC, 0.05% SDSsolution for 30 min and repeated once. Further washing was performed at50° C., in 0.1×SSC, 0.1% SDS solution for 30 min twice. Analysis of thehybridization was performed by exposure to an imaging plate (Fuji Photofilm Co. Ltd., Japan) for three hours using a bioimaging analyzerBAS2000 (Fuji Photo Film Co., Ltd., Japan). Northern blot techniqueanalysis was performed using probes used in Example 13 and MTN blot(CLONTECH, U.S.A.), so that the expression thereof in human wasexamined. As a result, the expression of mRNA at about 2.6 kb wasconfirmed in many organs including the liver, the placenta, the skeletalmuscle, and the uterus. In mouse, the expression of mRNA at about 2.6 kbwas confirmed in similar organs.

Example 15 Expression of Mouse SCR-1 in Stromal Cell

(1) Construction of Retrovirus Vector for Expression of Mouse SCR-1

Only ORF sequence in SCR-1 gene (a nucleotide sequence from nucleotidesnumbers 511 to 1350 in SEQ ID NO: 31) was inserted into a retrovirusvector, so that a vector for expression in stromal cells wasconstructed.

Messenger RNA was purified from one milligram of the total RNA inAGM-s3-A9 cell according to the protocol of the mRNA purification kit(Amersham Pharmacia, U.S.A.). cDNA was synthesized from this mRNAaccording to the conventional method. The following primers weresynthesized and PCR at 30 cycles was performed using the above describedcDNA as a template and Platinum Pfx DNA Polymerase (GIBCO Lifetech,U.S.A.), one cycle being a step performed at 94° C. for 20 seconds, at55° C. for 30 seconds, and 68° C. for one minute. m782F2:CCGCTCGAGCCACCATGCACTTGCGACTGATTTC (SEQ ID NO: 43) m782R2:ATTGAATTCCTAGTGTACAGTGCTGACTG (SEQ ID NO: 44)

An amplified fragment was digested with restriction enzymes EcoRI andXhoI. After electrophoresis, a DNA fragment was purified using JETSORB(Genomed, Germany). The purified DNA fragment was ligated withpMX-IRES-GFP vector digested with EcoRI and XhoI (gift form Professor T.Kitamura, TOKYO UNIV. INST. OF MEDICAL SCIENCE, Japan). The pMX-IRES-GFPvector was a plasmid in which IRES GFP was inserted into the retrovirusvector pMX. The obtained recombinant vector was transferred into E. ColiDH5a, and was seeded on LB agar medium containing 100 mg/ml ofampicillin, so that independent colonies were formed. After the isolatedcolony was cultured in 100 mL of LB medium containing 100 mg/ml ofampicillin, plasmid was purified using QIAGENtip100 (QIAGEN, U.S.A.).The sequence of the inserted gene was determined using conventionalmethod, so that the sequence was confirmed to be identical to thecorresponding region in SEQ ID NO: 31.

(2) Transfer of Mouse SCR-1 into Stromal Cell

Initially, BOSC23 cells at 2×10⁶ cells/dish were seeded on a collagentype I coated 60 mm dish (Asahi technoglass), and cultured in DMEMmedium containing 10% FCS at 37° C., under an atmosphere of 5% CO₂, andat a humidity of 100%. Twelve to 18 hours after the start of theculture, the medium was replaced by two milliliters of OPTI MEM medium(GIBCO BRL).

About 3 μg of plasmid inserted with SCR-1 into the above describedpMX-IRES-GFP was added to 18 μL of LIPOFECTAMINE Reagent (GIBCO BRL)diluted with 100 μL of OPTI MEM medium, and allowed to stand at roomtemperature for 30 min. The prepared DNA solution was added to theabove-prepared BOSC23 cell culture solution. After about five hours, twomilliliters of DMEM medium containing 20% FCS (GIBCO BRL) was added.

IRES (Internal Ribosome Entry Site) was determined by an access of theribosome to the internal site of the mRNA. Therefore, two genes could beexpressed from one mRNA caused by ligation of upward and downward genesseparated by IRES in one transcription unit during the construction ofan expression vector. With respect to the above-described plasmid, cDNAof SCR-1 was inserted in upward site and GFP (Green FluorescenceProtein) was inserted in downward site. Thus, the expression of SCR-1could be monitored by detecting the expression of GFP using FACS.

After about 24 hours, the medium was replaced by 4 ml of DMEM containing10% FCS. Further, after about 48 hours, the culture medium washarvested. After the culture medium was filtrated through 0.45 μmfilter, the filtrate was centrifuged at 1,200 g for 16 hours and thesupernatant was removed, so that the virus precipitation was obtained.

AGM-s3-A7 cells were cultured in one milliliter of MEMα mediumcontaining 10% FCS (GIBCO BRL) on a 24-well culture dish (FALCON) at1×10⁴ cells/well. After 12 to 18 hours, the virus precipitation wassuspended in one milliliter of MEMα medium containing 10% FCS, so thatthe stromal cell culture medium and the virus suspension were replaced.Next, POLYBRENE (Sigma, SEQUA-BRENE) was added to be 10 μg/mL. After theculture dish was centrifuged at 700 g for 45 min., the cells werecultured at 37° C., under an atmosphere of 5% CO₂, and at a humidity of100%. After 48 hours, the medium was replaced by one milliliter of MEMmedium containing 10% FCS. After 24 hours, the cells were passaged on a6-well culture dish (FALCON) and cultured in three milliliters of MEMmedium containing 10% FCS. Forty-eight hours after the passage, GFPexpression in the stromal cells was detected using a cell sorter(FACSVantage, Becton Dickinson), so that it was indirectly confirmedthat not less than 80% cells expressed SCR-1.

Example 16 Co-Culture of Stromal Cells in which SCR-1 Gene wasOverexpressed with Mouse Hematopoietic Stem Cells

AGM-s3-A9 cells, AGM-s3-A7 cells, or AGM-s3-A7 cells, transduced withSCR-1 gene by retrovirus infections, were seeded in a 24-well culturedish at a density of 1×10⁵ cells/well, and were cultured in MEMα mediumcontaining 10% FCS for one day in order to allow the cells toproliferate to cover the whole bottom surface of the culture dish.

Then, the medium was replaced by 1 ml of fresh medium and thirty cellsof the mouse hematopoietic stem cells (derived from C57BL/6-Ly5.1)obtained in Example 9 were sorted on this cell layer to initiate theco-cultures.

After 7 days of culture, all the cells in the co-culture were harvestedby trypsinization (0.05% trypsin in PBS containing 0.5 mM EDTA at 37° C.for two to five minutes), and the extent of the expansion ofhematopoietic stem and/or progenitor cells was analyzed in the followingexperiment.

Example 17

Transplantation of Hematopoietic Cells into Irradiated Recipient Mice

Thirty freshly isolated hematopoietic stem cells, obtained fromC57BL/6-Ly5.1 mice by the procedure described above (CD34 negative,Sca-1 positive, c-Kit positive, Lin negative cells, or cells derivedfrom C57BL/6-Ly5.1 pep mouse), or whole the cells harvested on the 7thday of the co-culture, which was initiated with 30 hematopoietic stemcells, were transplanted into the five C57BL/6-Ly5.2 mice (eight weeksage and male, Charles River), which were irradiated with X-ray at 8.5Gy, through the tail vein together with the 200,000 whole bone marrowcells derived from C57BL/6-Ly5.2 mice (Charles River).

After the transplantation, the peripheral blood cells were collectedfrom the retro-orbital sinus over time, and analyzed for the proportionof the cells that were derived from Ly5.1 hematopoietic cells. Theperipheral blood was analyzed according to the usual method (S. Kouzu,Fundamental techniques for immunology, YODOSHA, 1995). In order to lyseerythrocytes, three hundreds and fifty μL of distilled water was addedto 50 μL of the peripheral blood and 30 seconds after addition ofdistilled water, the same amount of the twice concentrated PBS andcentrifuged, so that white blood cells were recovered. After the cellswere washed once using the staining buffer (PBS containing 5% FCS and0.05% NaN₃), they were stained with anti-CD16 antibody, FITC-anti-Ly5.1,PE-anti-myeloid cells (Gr-1 and CD11c) and APC-anti-lymphoid cells (B220and Thy 1)(purchased from Pharmingen) and incubated for 30 min on ice.Stained cells were washed using the staining buffer and FACS analysiswas performed.

Expansions in the number of the cells, that were capable ofreconstituting hematopoietic cells during the culture of thehematopoietic stem cells were estimated by calculating proportions ofLy5.1 positive cells in the Gr-1 or CD11c positive cells (myeloidcells), or Ly5.1 positive cells in the CD90 or CD45R positive cells(lymphoid cells) in the peripheral blood in the transplanted mice atintervals.

FIG. 7 shows the results. When the cells co-cultured with AGM-s3-A7cells were transplanted, high chimerism derived from the cultured Ly5.1hematopoietic cells was not observed. From this result, it wasdemonstrated that AGM-s3-A7 cells themselves showed low activity tosupport the hematopoietic stem cells or hemopoietic precursor cells.When cells co-cultured with AGM-s3-A7 cells in which SCR-1 gene wasoverexpressed were transplanted, a significant high proportion of cellsderived from the cultured cells was detected in both myeloid andlymphoid cells in the peripheral blood. Therefore, it was clear that thehematopoietic stem cells and the hemopoietic precursor cells, whichcould reconstitute the hemopoietic system of the irradiated mice, weresupported and amplified on the A7 stromal cells in which SCR-1 gene wasoverepressed.

As a result, it was evident that SCR-1 had a function to give thestromal cells without an activity to support the survival or theproliferation of the hematopoietic stem cells or the hematopoieticprogenitor cells the above described activity. From these results, itwas evident that SCR-1 had an activity to effect on support theproliferation or the survival of the hematopoietic stem cells or thehemopoietic precursor cells; or an activity to effect the stromal cellsso as to give them an activity to support the hematopoietic stem cells.

Example 18 SCR-1 Transgenic Mice

The activity of mouse and human SCR-1 can be confirmed by establishinggenetically modified mice, such as transgenic mice. Appropriatepromoters are selected for expression of SEQ ID NOS: 31 and 33 whichallows their activity for hematopoietic stem cell growth or survivalpromotion to be confirmed. GATA-2 promoter drives expression of genes invery early hematopoietic stem or progenitor cell population. Expressionof the SCR-1 gene (SEQ ID NOSL 31 or 33) under the regulation of GATA-2promoter in transgenic mice will cause the hematopoietic stem orprogenitor cells to express the SCR-1 gene. This SCR-1 gene expressionin hematopoietic stem progenitor cells will lead to expansion ofhematopoietic stem or progenitor cells which will result in an increaseof hematopoietic cells in embryos, neonates or adult mutant mice. GATA-2promoters described by Minegishi et al may be used for this purpose (J.Biol. Chem. 1998 Feb. 6;273(6):3625-34).

The SCR-1 gene (SEQ ID NOS: 31 or 33) may also be expressed under thecontrol of CAG or other promoters that work in ubiquitous tissues(Kiwaki et al. Gene Ther, 1996 May 1;7(7):821-30). This will allow fordetermination of the effects of SCR-1 gene expression in other tissuecell types together with hematopoietic cells. Transgenic mice can beestablished accroding to the methods described in “Manipulating theMouse Embryo” (Brigid Hogan, Rosa Beddington, Frank Costantini,Elizabeth Lacy, 1994, Cold Spring Harbor Laboratory Press).

1. An isolated polynucleotide encoding a polypeptide having stem cellgrowth factor activity, said polynucleotide comprising the nucleotidesequence of SEQ ID NO: 9, 11, 12, 31 or 33 or the mature protein codingportion thereof, or a fragment, analog, variant or derivative thereofthat encodes a polypeptide retaining stem cell growth factor activity.2. The polynucleotide of claim 1 which hybridizes to the complement ofthe nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or 33 understringent hybridization conditions.
 3. The polynucleotide of claim 1which comprises a nucleotide sequence having greater than about 85%sequence identity with the nucleotide sequence of SEQ ID NO:9, 11, 12,31 or
 33. 4. The polynucleotide of claim 1 which comprises a nucleotidesequence having greater than about 90% sequence identity with thenucleotide sequence of SEQ ID NO:9, 11, 12, 31 or
 33. 5. Thepolynucleotide of claim 3 comprising a nucleotide sequence havinggreater than about 92% sequence identity with the nucleotide sequence ofSEQ ID NO:9, 11, 12, 31 or
 33. 6. The polynucleotide sequence of any ofclaims 1, 2 or 3 with the proviso that said polynucleotide sequence doesnot consist of the nucleotide sequence of SEQ ID NO:
 47. 7. An isolatedpolynucleotide that comprises the mature protein coding sequence of SEQID NO: 9, 11, 12, 31 or
 33. 8. An isolated polynucleotide that comprisesthe nucleotide sequence of SEQ ID NO: 9, 11, 12, 31 or
 33. 9. The DNA ofclaim 1 encoding: (A) a polypeptide which has an amino acid sequencecomprising at least amino acid residues 22 to 279 of SEQ D NO: 32, or anamino acid sequence comprising at least amino acid residues 22 to 272 ofSEQ ID NO: 34; or (B) a polypeptide which has an amino acid sequenceincluding deletion, substitution or insertion of one or several aminoacids in the amino acid sequence comprising at least amino acid residues22 to 279 of SEQ ID NO: 32, or an amino acid sequence comprising atleast amino acid residues 22 to 272 of SEQ ID NO: 34, and which has anactivity to support proliferation or survival of hematopoietic stem cellor hematopoietic progenitor cell, with a proviso that C-terminal aminoacid sequence does not comprise the amino acid sequence of SEQ ID NO:46.
 10. The DNA according to claim 1, which is: (a) a DNA whichcomprises at least nucleotides 574 to 1347 of SEQ ID NO: 31; or (b) aDNA which is hybridizable with the nucleotide sequence of SEQ ID NO: 31or a probe prepared from said sequence, under stringent conditions, andwhich has an activity to support proliferation or survival ofhematopoietic stem cell or hematopoietic progenitor cell.
 11. The DNAaccording to claim 10, wherein the stringent conditions are 6×SSC 5×Denhardt, 0.5% SDS and 68° C. (SSC 3M NaCl, 0.3M sodium citrate, 50×Denhardt 1% BSA/1% polyvinyl pyrrolidone, 1% Ficoll 400, or 6×SSC, 5×Denhardt, 0.5% SDS, 50% formamide and 42° C.
 12. The DNA according toclaim 1, which is: (a) a DNA which comprises at least nucleotides 321 to1074 of SEQ ID NO: 33; or (b) a DNA which is hybridizable with thenucleotide sequence of SEQ ID NO: 33 or a prove prepared from saidsequence, under stringent conditions, and which has an activity tosupport proliferation or survival of hematopoietic stem cell orhematopoietic progenitor cell.
 13. The DNA according to claim 12,wherein the stringent conditions are 6×SSC/5× Denhardt, 0.5% SDS and 68°C. (SSC 3M NaCl, 0.3M sodium citrate, 50× Denhardt/1% BSA/1% polyvinylpyrrolidone, 1% Ficoll 400, or 6×SSC, 5× Denhardt, 0.5% SDS, 50%Formamide and 42° C.
 14. The polynucleotide of any of claims 1, 2 or 3which is a DNA.
 15. An isolated polynucleotide which comprises thecomplement of the polynucleotide of any one of claims 1, 2 or
 3. 16. Avector comprising the polynucleotide of any one of claims 1, 2 or
 3. 17.An expression vector comprising the polynucleotide any one of claims 1,2 or
 3. 18. A host cell genetically engineered to express thepolynucleotide of any one of claims 1, 2 or
 3. 19. A host cellgenetically engineered to contain the polynucleotide of any one ofclaims 1, 2 or 3 in operative association with a regulatory sequencethat controls expression of the polynucleotide in the host cell.
 20. Thehost cell of claim 19 which has been genetically engineered to contain aheterologous regulatory sequence that increases expression of anendogenous polynucleotide.
 21. A method of producing a polypeptidehaving stem cell growth factor activity comprising growing the host cellof claim 19 in a culture medium under conditions that permit expressionof said polypeptide and isolating said polypeptide from said host cellor said culture medium.
 22. A polypeptide produced by the method ofclaim
 21. 23. An isolated polypeptide comprising the amino acid sequenceof SEQ ID NO: 10, 13, 16, 32 or 34, or the mature protein portionthereof, or a fragment, analog, variant or derivative thereof thatretains stem cell growth factor activity.
 24. The polypeptide of claim23 which is encoded by a polynucleotide of claim
 2. 25. The polypeptideof claim 23 which comprises an amino acid sequence having greater thanabout 85% sequence identity with the nucleotide sequence of SEQ ID NO:10, 13, 16, 32 or
 34. 26. The polypeptide of claim 23 which comprises anamino acid sequence having greater than about 92% sequence identity withthe nucleotide sequence of SEQ ID NO: 10, 13, 16, 32 or
 34. 27. Thepolypeptide of claim 23 with the proviso that said polypeptide does notconsist of the amino acid sequence of SEQ ID NO:
 48. 28. An isolatedpolypeptide comprising the mature protein portion of SEQ ID NO: 10, 13,16, 32 or
 34. 29. The polypeptide of claim 23, wherein the polypeptidecomprises one or more motifs selected from the group of a laminin-typeEGF-like domain, a membrane attack complex component/perforin domain,and neurohypophysial hormone signature.
 30. A polypeptide which is anexpression product of a polynucleotide according to any one of claims1-3, the polypeptide having an activity to support proliferation orsurvival of hematopoietic stem cell or hematopoietic progenitor cell,with a proviso that C-terminal amino acid sequence does not comprise theamino acid sequence of SEQ ID NO:
 46. 31. The polypeptide according toclaim 8, which has an amino acid sequence comprising at least amino acidresidues 22 to 279 of SEQ ID NO: 32, or an amino acid sequence includingdeletion, substitution or insertion of one or several amino acids in theamino acid sequence comprising at least amino acid residues 22 to 279 ofSEQ ID NO
 32. 32. The polypeptide according to claim 23, which has anamino acid sequence comprising at least amino acid residues 22 to 272 ofSEQ ID NO: 34, or an amino acid: sequence including deletion,substitution or insertion of one or several amino acids in the aminoacid sequence comprising at least amino acid residues 22 to 272 of SEQID NO:
 34. 33. The polypeptide according to claim 23, which is modifiedwith one or more modifying agent selected from the group consisting ofpolyethylene glycol (PEG), dextran, poly(N-vinyl-pyrrolidone),polypropylene glycol homopolymer, copolymer of polypropyleneoxide/ethylene oxide, polyoxyethylated polyol and polyvinyl alcohol. 34.The polypeptide of claim 23 comprising at least ten consecutive aminoacids from SEQ ID NO: 10 or
 13. 35. The polypeptide of claim 23,comprising at least ten consecutive amino acids from the C-terminalseventeen amino acids of SEQ ID NO: 10 or
 13. 36. A polypeptide withbiological activity, said polypeptide comprising at least 272 aminoacids and having at least 98% identity with SEQ ID NO:
 10. 37. Anisolated polypeptide with stem cell growth factor activity having atleast 90% identity with SEQ ID NO: 10, 13, or 16 and lacking amino acidsequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29) wherein A=Alanine,C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine,G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine,N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine,V=Valine, W=Tryptophan, Y=Tyrosine.
 38. An isolated polypeptide withstem cell growth factor activity having at least 90% identity with SEQID NO: 10, 13, or 16 and lacking any 10 consecutive amino acids from aamino acid sequence GIEVTLAEGLTSVSQRTQPTPCRRRYL (SEQ ID NO: 29), whereinA=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid,F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.39. An isolated polypeptide with stem cell growth factor activity havingat least an amino acid sequence SVSVSTVH (SEQ ID NO: 27) or VSVSTVH (SEQID NO: 28), wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H-Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.40. (canceled)
 41. A kit comprising the polypeptide of claim
 23. 42. Aculture medium comprising an amount of the polypeptide of claim 23effective to maintain survival of or promote proliferation of a stemcell or germ cell.
 43. A composition comprising the polypeptide of claim23 and a pharmaceutically acceptable carrier or diluent.
 44. Thecomposition of claim 43 that is a pharmaceutical composition.
 45. Thepharmaceutical composition of claim 44 having an effect to supportproliferation or survival of hematopoietic stem cell or hematopoieticprogenitor cell, which comprises: (A) a polypeptide which has an aminoacid sequence comprising at least amino acid residues 22 to 279 of SEQID NO: 32, or an amino acid sequence comprising at least amino acidresidues 22 to 272 of SEQ ID NO: 34; or (B) a polypeptide which has anamino acid sequence including deletion, substitution or insertion of oneor several amino acids in the amino acid sequence comprising at leastamino acid residues 22 to 279 of SEQ ID NO: 32; or an amino acidsequence comprising at least amino acid residues-22 to 272 of SEQ ID NO:34, and which has an activity to support proliferation or survival ofhematopoietic stem cell or hematopoietic progenitor cell.
 46. Anantibody that binds to the polypeptide of claim
 23. 47. The antibody ofclaim 46 that specifically binds to a polypeptide having the amino acidsequence of SEQ ID NO: 10, 13, 16, 32 or
 34. 48. The antibody of claim46 that does not bind to a polypeptide having the amino acid sequence ofSEQ ID NO:
 48. 49. The antibody of claim 46 that is a polyclonalantibody, monoclonal antibody, antibody fragment, chimeric antibody, orhumanized antibody.
 50. A kit comprising the antibody of claim
 46. 51. Amethod for detecting the polynucleotide of claim 1 in a sample,comprising: a) contacting the sample with a compound that binds to andforms a complex with the polynucleotide for a period sufficient to formthe complex; and b) detecting the complex, so that if a complex isdetected, the polynucleotide is detected.
 52. A method for detecting thepolynucleotide of claim 1 in a sample, comprising: a) contacting thesample under stringent hybridization conditions with nucleic acidprimers that anneal to the polynucleotide under such conditions; b)amplifying a product comprising at least a portion of thepolynucleotide; and c) detecting said product and thereby thepolynucleotide in the sample.
 53. canceled
 54. A method for detectingthe polypeptide of claim 23 in a sample, comprising: a) contacting thesample with a compound that binds to and forms a complex with thepolypeptide under conditions and for a period sufficient to form thecomplex; and b) detecting formation of the complex, so that if a complexformation is detected, the polypeptide is detected.
 55. A method foridentifying a compound that binds to the polypeptide of claim 23,comprising: a) contacting the compound with the polypeptide underconditions and for a time sufficient to form a polypeptide/compoundcomplex; and. b) detecting the complex, so that if thepolypeptide/compound complex is detected, a compound that binds to thepolypeptide is identified.
 56. A method for identifying a compound thatbinds to the polypeptide of claim 23, comprising: a) contacting thecompound with the polypeptide, in a cell, for a time sufficient to forma polypeptide/compound complex, wherein the complex drives expression ofa reporter gene sequence in the cell; and b) detecting the complex bydetecting reporter gene sequence expression, so that if thepolypeptide/compound complex is detected, a compound that binds to thepolypeptide is identified.
 57. A nucleic acid array comprising thepolynucleotide of claim 1 or a unique segment of the polynucleotide ofclaim 1 attached to a surface.
 58. The array of claim 57, wherein thearray detects full-matches to the polynucleotide or a unique segment ofthe polynucleotide of claim
 1. 59. The array of claim 57, wherein thearray detects mismatches to the: polynucleotide or a unique segment ofthe polynucleotide of claim
 1. 60. A method of treatment of a subject inneed of enhanced activity or expression of stem cell growth factor-likepolypeptide of claim 23 comprising administering to the subject (a) acomposition comprising a therapeutic amount of an agonist of saidpolypeptide; (b) a composition comprising a therapeutic amount of thepolypeptide; or (c) a composition comprising a therapeutic amount of apolynucleotide encoding the polypeptide in form and under conditionssuch that the polypeptide is produced; said composition comprising apharmaceutically acceptable carrier or diluent.
 61. A method oftreatment of a subject having need of decreased activity or expressionof stem cell growth factor-like polypeptide of claim 23, comprisingadministering to the subject: (a) a composition comprising a therapeuticamount of an antagonist of said polypeptide; (b) a compositioncomprising a therapeutic amount of the polynucleotide that inhibits theexpression of the nucleotide sequence encoding said polypeptide; and (c)a composition comprising a therapeutic amount of a polypeptide thatcompetes with the stem cell growth factor-like polypeptide for itsligand; said composition comprising a pharmaceutically acceptablecarrier or diluent. 62-65. canceled
 66. A stromal cell geneticallyengineered to express a polypeptide of claim 23 in an amount effectiveto support proliferation or survival of a stem cell or germ cell. 67.The stromal cell of claim 66 wherein said cell is a primordial germcell, germ line stem cell, embryonic stem cell, hematopoietic stem cell,hematopoietic progenitor cell, pluripotent cell, or totipotent cell. 68.An implant comprising a cell genetically engineered to express apolypeptide of claim 23 in an amount effective to support proliferationor survival of a stem cell or germ cell.
 69. The implant of claim 68wherein said cell is a primordial germ cell, germ line stem cell,embryonic stem cell, hematopoietic stem cell hematopoietic progenitorcell pluripotent cell, or totipotent cell.
 70. An isolatedpolynucleotide comprising the protein coding cDNA insert of the plasmiddeposited with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology Zip code305-8566; Higashi 1-1-3, Tsukuba, Ibaraki, Japan) on Jun. 26, 2000 underaccession number FERM BP-7198.
 72. The mature polypeptide expressed bythe polynucleotide of claim 70 in a suitable host cell.
 73. An isolatedpolynucleotide comprising the protein coding cDNA insert of the plasmiddeposited with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (Zip code305-8566; Higashi 1-1-3, Tsukuba, Ibaraki, Japan) on Jun. 26, 2000 underaccession number FERM BP-7197.
 74. The mature polypeptide expressionproduct expressed by the polynucleotide of claim 73 in a suitable hostcell.